EPA-600/3-77-019
February 1977
Ecological Research Series
ACUTE AND CHRONIC TOXICITY OF
CHLORDANE TO FISH AND
INVERTEBRATES
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-019
February 1977
ACUTE AND CHRONIC TOXICITY OF
CHLORDANE TO FISH AND INVERTEBRATES
by
Rick D. Cardwell
Dallas G. Foreman
Thomas R. Payne
Doris J. Wilbur
Chemico Process Plants Company - Envirogenics Systems
El Monte, California 91734
Contract No. 68-01-0187
D. T. Allison
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY - DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory - Duluth, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation
for use.
11
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FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water—for recreation, food, energy, transportation, and
industry—physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in
water. The Environmental Research Laboratory in Duluth, Minnesota, develops
methods, conducts laboratory and field studies, and extrapolates research
findings.
--to determine how physical and chemical pollution affects aquatic
life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through use of
models
—to measure bioaccumulation of pollutants in aquatic organisms that
are consumed by other animals, including man
This report describes the acute and chronic effects of the pesticide
chlordane on a number of freshwater fishes and invertebrates.
Donald I. Mount, Ph.D.
Di rector
Environmental Research Laboratory
Duluth, Minnesota
111
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ABSTRACT
The acute and chronic toxicity of technical chlordane to bluegill
(Lepomis macrochirus), fathead minnow (Pimephales promelas), brook trout
(Sa1veli nu s fontinaTis), Daphnia magna, Hyallela azteca, and Chironomus
No. 51 were determined with flow-through conditions^The purpose was to
estimate concentrations producing acute mortality and those haying no effect
on the long-term survival, growth, and reproduction of the various species.
Whole body residues of technical chlordane components were measured in the
three invertebrate species at the end of the chronic exposure tests.
Concentrations of technical chlordane causing 50% mortality in 96 hr
were 36.9 yg/1 for fathead minnow, 47 yg/1 for brook trout, and 59 yg/1 for
bluegill, while that causing 50% immobilization in the cladoceran, p.. magna.
was 28.4 yg/1. The amphipod, H. azteca, was only slightly affected at 96 hr
by the chlordane concentrations tested, and the 168-hr EC50 was 97.1 yg/1.
Acute mortality of midges, Chironomus No. 51, was not successfully evaluated.
With respect to the test conditions employed and life cycle stages
evaluated, the lowest concentrations of technical chlordane found to cause
major chronic effects were 0.32 yg/1 for brook trout, 1.22 yg/1 for bluegill,
1.7 yg/1 for midges, 11.5 yg/1 for amphipods, and 21.6 yg/1 for cladocerans.
Technical chlordane accumulation in the invertebrate species varied
directly with the aqueous concentration to which the animals were exposed.
The component accumulated to the greatest extent was trans-nonachlor, for
which whole body residues were up to 145,000-times higher than the aqueous
concentration.
This report was submitted in fulfillment of Contract No. 68-01-0187
by the Chemico Process Plants Company-Envirogenics Systems under the sponsor-
ship of the U.S. Environmental Protection Agency. Work was completed as of
June 1974.
IV
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CONTENTS
Foreword iii
Abstract iv
Tables vi
List of Abbreviations and Symbols ix
Acknowledgments x
I Introduction 1
II Conclusions 3
III Recommendations 4
IV Literature Review 6
V Materials and Methods 21
VI Results 34
VII Discussion 76
Literature Cited 81
Bibliography 87
Appendix Tables 90
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TABLES
No. Page
1 Concentrations of Chlordane Toxic to
Fish 10
2 Concentrations of Chlordane Toxic to
Aquatic Invertebrates 16
3 Characteristics of Fish Exposed to
Technical Chlordane in Acute Toxicity
Tests 22
4 Water Quality During Acute Toxicity
Tests of Technical Chlordane 35
5 Measured Concentrations of Technical
Chlordane in Acute Toxicity Tests 37
6 Total Lengths of Fathead Minnow Fry
Chronically Exposed to Technical
Chlordane 40
7 Lengths and Weights of Adult Fathead
Minnows at Termination of Chronic
Exposure to Technical Chlordane 41
8 Mortality of F -Generation Fathead
Minnows During Chronic Exposure to
Chlordane 43
9 Spawning History of Fathead Minnows
Chronically Exposed to Technical
Chlordane 44
10 Mortality and Relative Size of F,-
Generation Fathead Minnows Chronically
Exposed to Technical Chlordane 45
11 Growth of F -Generation Bluegill
During Chronic Exposure to Technical
Chlordane 47
VI
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No. Page
12 Mortality of F -Generation Bluegill
During Chronic0Exposure to Technical
Chlordane 49
13 Spawning History of Bluegill Chronically
Exposed to Technical Chlordane 50
14 Conditions of Adult Bluegill at
Termination of Chronic Toxicity Test 52
15 Survival of F-,-Generation Bluegill
in Chronic Toxicity Test of Technical
Chi ordane 54
16 Growth of F,-Generation Bluegill
During Chronic Toxicity Test of
Technical Chlordane 55
17 Total Lengths of F -Generation Brook
Trout Chronically Exposed to Technical
Chi ordane 57
18 Body Weights of F -Generation Brook
Trout Chronically Exposed to Technical
Chlordane 58
19 Mortality of F -Generation Brook Trout
Chronically Exposed to Technical
Chlordane 59
20 Spawning Success of Brook Trout
Chronically Exposed to Technical
Chi ordane 60
21 Viability and Hatch of Embryos and
Conditions of Fj-Generation Brook
Trout Alevins 61
22 Growth of F,-Generation Brook Trout
During Chronic Exposure to Technical
Chi ordane 63
23 Relative Survival and Growth of
Myall ela azteca Exposed to Technical
Chlordane 65
24 Contents and Concentration Factors (C.F.)
of Chlordane Constituents in Dried Hyallela
azteca That Had Been Exposed to Technical
Chlordane 67
vii
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No.
25 Survival and Reproduction of Daphnia
magna in Chronic Toxicity Testo?
Technical Chlordane 69
26 Average Dry Body Weights of First
Instar Daphnia magna Produced During
Fourth Week of Chronic Toxic ity Test
of Technical Chlordane 71
27 Contents and Concentration Factors (C.F.)
of Chlordane Constituents in Dried Daphnia
magna That Had Been Exposed to Technical
Chlordane 73
28 Chronic Effects of Technical Chlordane on
Chironomus No. 51 74
Vlll
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
LC50
EC50
MATC
A.I.
LCI 00
LCO
LT50
C.F.
SYMBOLS
S
mg/1
ug/g
ppb
median lethal concentration
median effective concentration
maximum acceptable toxicant concentration
active ingredient
lethal concentration to all test organisms
lethal threshold concentration
median lethal time
concentration factor
logarithm of the standard deviation of the population
tolerance frequency distribution
antilogarithm of a
milligram per liter
microgram per gram
parts per billion = microgram per kilogram or micro-
gram per liter
IX
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ACKNOWLEDGMENTS
We wish to thank Mr. D. T. Allison, Project Officer, for providing
valuable guidance during the course of the project and for critically review-
ing the manuscript. Mr. L. H. Mueller, Research Chemist at the Environmental
Research Laboratory, Duluth, Minnesota (ERL-D), provided valuable assistance
in analytical procedures for measuring chlordane in water and biological
tissues. Mr. W. E. Wright and Ms. J. L. Wright conducted all chemical anal-
yses of water quality and assisted in the conduct of the toxicity tests. Mr.
R. Stankiewicz contributed to all aspects of computer programming and anal-
ysis. Mr. W. Richardson of the California Department of Fish and Game
arranged for the acquisition of brook trout. Mr. K. E. Biesinger and Ms. B.
J. Halligan, ERL-D, provided helpful advice on culture and testing of daph-
nids and amphipods, respectively. Dr. M. Mulla, University of California
Department of Entomology (Riverside), supplied Chironomus No. 51 and sug-
gested effective culture techniques- We would also like to thank Ms. B.
Leistikow, Fisheries Research Institute, University of Washington, for pro-
viding positive identification of the amphipod, Myall el a azteca (Saussure).
Finally, we extend our appreciation to the Velsicol Chemical Corporation
(Chicago, Illinois) for supplying analytical reference technical chlordane
and literature on its composition for use in this program.
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SECTION I
INTRODUCTION
The organochlorine insecticide, chlordane, is widely used for the con-
trol of insect pests, particularly in non-agricultural areas. The insecti-
cide has been extensively used relative to other organochlorine insecticides;
in 1971, over 11.4 x 10 kg of chlordane was produced compared tOg5 x 10
kg endrin, dieldrin and lindane, 4.5 x 10 kg aldrin, and 21 x 10 kg DDT
(l). Because this chemical has a high biological potency and is relatively
long-lived in the environment (2), it presents a potential hazard to non-
target species (fish and wildlife) and ultimately to public health.
The adverse effects of chlordane on fish and aquatic invertebrates
can be examined with acute and chronic toxicity tests or with studies of
the accumulation of toxic (to predatory animals including man) residues
(3). All of these methods have limitations (4), but the chronic toxicity
test, which encompasses all or most of one reproductive cycle, probably
represents the most direct method of estimating the concentration of chlor-
dane which is "safe" for long-term survival, growth, and production of a
species.
Less than a decade ago, water quality criteria for aquatic organisms
were set with application factors ranging from 1/10 to 1/100 or with various
equations (5, 6). These factors were multiplied by an acute toxicity test
result such as the concentration lethal to 50% of the test specimens (LC50)
to estimate environmentally "safe" concentrations. But these application
factors were arbitrary and presented the hazard of over or underestimating
the "safe" level. Because of the need to adequately protect the aquatic
resource with realistic standards, other methods were examined. The chronic
toxicity test has been suggested as the most practicable tool for achieving
objective evaluations of sublethal, long-term toxicant effects on many spe-
cies. The concept of the chronic test, its methods, scope, and application,
was initially set forth by Mount and Stephan (7) in their studies of malation
and the butoxyethanol ester of 2,4-D using fathead minnows (Pimephales
prpmelas Rafinesque). Basically, the chronic test attempts to estimate the
toxicant concentration at which effects on survival, growth and reproduction
of all life stages become statistically indistinguishable from those for fish
held in uncontaminated water (controls). Between the concentration producing
some effect on one or more of the above indices and that having no effect
is the maximum acceptable toxicant concentration (MATC), the theoretical
"just safe" level. By dividing the MATC estimate by the LC50, an applica-
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tion factor can be obtained which can be used to estimate "safe" levels
for aquatic organisms which are unsuitable for chronic toxicity testing
for one reason or another. Several chronic toxicity tests of pesticides
have been completed since that of Mount and Stephan (7). These include
evaluations of malathion (8), carbaryl (9), and captan (10). Although
application factors may remain relatively constant for a particular chemical
and taxonomic group of organisms, the constraints on its applicability need
to be defined. It has been recently shown, that the MATC and application
factors can be satisfactorily estimated for at least one heavy metal through
the use of one rather than a multiple generation test (11).
As discussed in the next section, a moderate amount of information
is already available on the toxicity of chlordane to aquatic life. However,
the majority of the tests have been conducted with static conditions for
short periods and without measurement of chlordane concentrations in the
diluent water. While these tests suffice to define the approximate order of
chlordane toxicity to aquatic biota, their limited experimental scope re-
strains, in most cases, their use in establishing water quality criteria. To
our knowledge, no chronic toxicity, reproductive studies of chlordane have
heretofore been reported in the literature, and these studies are generally
thought to be necessary for establishing sound standards. Accordingly, this
investigation sought to define the acute and chronic toxicity of chlordane to
three species of freshwater fish and three invertebrates using flow-through
conditions. Furthermore, measurements of chlordane residues in tissues of
the chronically exposed invertebrates were incorporated into the experimental
design to provide information on the extent of bioaccumulation and on the
potential hazard to predator species from consuming animals contaminated
with this insecticide.
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SECTION II
CONCLUSIONS
1. The acute mortality tests suggested that technical chlordane was a
cumulative poison, causing toxicity as a function of concentration and
exposure time. Median lethal thresholds were not attained within 96 hr
for any species. The fathead minnow was the only species for which a
threshold was observed, and it did not occur until approximately 180 hr.
2. Technical chlordane was generally more toxic on a chronic sublethal
basis to the three fish species than to the three species of inverte-
brates.
3. Chronic toxicity test results suggest that technical chlordane concen-
trations greater than approximately 0.3 ug/1 would be deleterious to the
production of at least some fish species, and that concentrations greater
than 21.6 yg/1 would probably be very deleterious to most aquatic ani-
mals.
4. Accumulation of technical chlordane in the cladoceran and the amphipod
was substantial, but varied with respect to the component. Cis-nonachlor
was concentrated to the greatest extent and heptachlor the least. Resi-
dues of technical chlordane were not detected in the midge.
5. Measured concentrations of technical chlordane were consistently less
than desired, even though low concentrations of the non-ionic surfactant,
Triton X-100, and of the solvent, acetone, were employed to aid dis-
solution, and the toxicant solutions were continuously replenished.
This indicates that toxicity tests of this insecticide would not be
valid unless based upon measured concentrations of the dissolved
compound.
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SECTION III
RECOMMENDATIONS
1. Acute toxicity tests of technical chlordane should be continued until a
median lethal threshold is observed rather than discontinued at a speci-
fied time. This would permit characterization of the toxicity curve
particular to each species and life stage, and might prove useful in
calculating effects in mixing zones.
2. Additional acute and chronic toxicity tests of technical chlordane
using additional species of freshwater fish (e.g. Ictaluridae, Catasto-
midae), marine fish (e.g. Cyprinodontidae, Clupeidae), freshwater and
marine invertebrates, and algae are needed, regardless of whether water
quality standards are set for groups of aquatic organisms inhabiting
specific ecosystems or on the average MATC of the most sensitive groups.
Such information is needed to permit objective decisions on what levels
of this insecticide will have no effect on diverse communities of aquatic
organisms since there may or may not be important differences between
taxonomic groups and between freshwater and marine organisms.
3. The acute and chronic toxicities of technical chlordane in mixtures
of other toxicants and with different environmental conditions should be
determined to evaluate interactions.
4. A multiple generation chronic toxicity test should be conducted to
determine whether this complex insecticide causes teratogenic or muta-
genic effects. For freshwater fish a species which completes its life
cycle rapidly, such as the flagfish (Jordanella floridae Goode and Bean)
or a poeciliid (e.g. the mosquitofish, Gambusia affinis [Baird and
Girard]), should be considered since they have relatively short genera-
tion times of 3 to 4 months.
5. Studies of technical chlordane contents in fish and invertebrates should
incorporate investigations of the uptake, biotransformation, and tissue
distribution of each of the major, and perhaps the minor, constituents
(e.g. hexachlorocyclopentadiene).
7. The degree and efficiency of transfer of technical chlordane components
through several trophic levels should be determined and compared to
uptake from the water.
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8. The dosage levels of technical chlordane causing lethal and sublethal
effects on predators should be determined by feeding them prey species
contaminated with known amounts of the insecticide.
9. Increasing the number of replicates per treatment for the F -generation
from the presently recommended two to at least three and possibly four
would considerably strengthen the value and power of statistical tests.
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SECTION IV
LITERATURE REVIEW
CHEMISTRY OF CHLORDANE
Chlordane (1, 2, 4, 5, 6, 7, 8, 8-octachloro-2, 3, 3a, 4, 7, 7a-hexa-
hydro-4, 7-methanoindene) is a chlorinated hydrocarbon insecticide manufac-
tured by the Velsicol Chemical Corporation (Chicago).
Cl
The technical grade used in the present program was supplied by Velsicol
and identified as "Analytical Reference Technical Chlordane." It is a com-
plex mixture and has been variously characterized by Velsicol (12), the U.S.
Environmental Protection Agency (EPA, 13), and Saha and Lee (14). The pre-
dominant constituents are trans-chlordane (24 +_ 2%), cis_-chl ordane (19 ±
3%), heptachlor (10 +_3%), chlordenes (20.5%), trans-nonachlor (5.1%), and
cis,-nonachlor (2.8%) (page 7 and Appendix Table
Technical Chlordane is a liquid with a molecular weight of approximately
410. Its solubility limit in the laboratory water used in these investiga-
tions was of the order of 150 to 220 yg/1 at 22°C. Edwards (15) has given
its solubility as 100 yg/1 at 20 to 30°C. A typical chromatogram of the
technical material is shown in Fig. 1. Identification was accomplished with
known standards and EPA (13) data.
The stability of technical Chlordane solutions in distilled water has
been evaluated by Bevenue and Yeo (16) for a period of 60 days. The authors
-------
Cl,
Cl
trans-chlordane
cis-chlordane
heptachlor
nonachlor
chlordene
Cl '.
Cl
cis-nonachlor trans-nonachlor
chlordene
-------
oo
Fig. 1. Chromatogram of technical chlordane standard in hexane run under
conditions specified elsewhere: (1) n-hexane; (2) ClnHfiClfi isomer-,
(3) heptachlor and chlordene; (4) 7 - @ and "A"-chlordener (5) C,0H7
isomer; (6) trans-chlordane; (7) c_^s_-chlordane; (8) trans-nonachlorj
(9) cis-nonachlor.
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noted no temporal changes in trans- (y) chlordane and cis- (a) chlordane, or
the nonachlor components, but observed a conversion of heptachlor to 1-hydroxy-
chlordene. There was no generation of heptachlor epoxide, and the occurrence
of oxychlordane was not mentioned. Octachlor epoxide, also known as oxychlor-
dane, is formed by the epoxidation of chlordane and is a known metabolite of
chlordane in animals (17, 18).
TOXICITY OF CHLORDANE TO AQUATIC ANIMALS
Although chlordane has received far less study than such chlorinated
hydrocarbons as DDT and endrin, a substantial volume of literature exists
concerning its toxicity to aquatic animals. The majority of the literature
concerns the insecticide's efficacy in killing mosquitoes (Diptera: Cul-
icidae).
Fish
Chlordane is generally less acutely toxic to fish than endrin, DDT,
dieldrin, and aldrin, and more toxic than lindane and methoxychlor, for
example. Much of the available literature on chlordane toxicity to fish
is summarized in Table 1. The majority of these toxicity tests were con-
ducted for less than 96 hr and, except for two, employed static test con-
ditions. In preparing Tables 1 and 2, efforts were made to convert test
results, where possible, to concentrations equivalent to 100% active ingre-
dient (A.I.) for direct comparison. As can be seen, responses to chlordane
ranged from 0.1 ug/1 A.I., which significantly increased oxygen consumption
in bluegill (19) to 3,050 yg/1 A.I., a level lethal to rainbow trout exposed
to an emulsion of chlordane in a flow-through system (20).
Comparison of the toxicity test results conducted with methodology
similar or identical to the standard procedures set forth by Doudoroff et al.
(5) and the American Public Health Association (APHA, 21) lessens the dis-
parity in reported toxic concentrations. Henderson, Pickering and Tarzwell
(22) exposed four species of freshwater fish to an enulsifiable concentrate
of 75% chlordane and found 96-hr LC50 values which varied from 16.5 yg/1
(bluegill) to 142.5 yg/1 (guppy, Poecilia reticulata). The concentrate was
also more toxic to fathead minnows in soft water than in hard. During the
same year, Clemens and Sneed (24) reported that 500 yg/1 chlordane produced
50% mortality in channel catfish (Ictalurus punctatus) finger!ings in 96 hr.
The later data of Katz (23) for three species of salmonids and the euryhaline
stickleback (Gasterosteus aculeatus) and of Macek, Hutchinson, and Cope (25)
for bluegill were in closer aggreement with the data of Henderson et al. (22)
than with that of Clemens and Sneed (24). Ninety-six hour median lethal
concentrations for the three salmonids ranged from 44 to 57 yg/1 (23). The
study of Macek et al. (25) indicated that chlordane was more toxic to blue-
gill at higher water temperatures than at lower ones. At higher temperatures
successively less chlordane was required to produce 50% mortality in 24 hr.
After 96 hr bluegill were killed less rapidly at 12.7°C (LC50 of 85 yg/1)
than at 18.3°C (LC50 of 70 yg/1), but they were killed at the same concen-
tration between 18.3°C and 23.8 °C.
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TABLE 1. CONCENTRATIONS OF CHLORDANE TOXIC TO FISH
Species
Common
Chinook salmon
Coho salmon
Rainbow trout
Threesplne
stickleback
Threesplne
stickleback
Fathead minnow
Fathead minnow
Bluegill
Goldfish
name
Binomial
Oncprhyncnus
tsWwytscha
0. kisutch
Salmo ciairdneri
Gasterosteus
aculeatus
Gasterosteus
aculeatus
Pimephales promelas
Pimephales promelas
Lepomis macrochirus
Carassius auratus
Response manifest
at
time,
hr
96
96
96
96
96
96
96
96
96
a
cone.,
ug/1
57.0
56.0
44.0
90C
160d
39e
52f
16.5
61.5
Type of
response
LC50b
LC50
LC50
LC50
LC50
LC50
LC50
LC50
LC50
Reference
23
23
23
23
23
22
22
22
22
Continued
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TABLE 1. CONCENTRATIONS OF CHLORDANE TOXIC TO FISH—continued
Species name
Common
Guppy
Channel catfish
Bluegill
Bluegill
Murrel
Murrel
Murrel
Rohu
Spiny eel
Binomial
Poecilia reticulata
Ictalurus punctatus
L, macrochirus
L. macrochirus
Channa punctatus fry
Channa punctatus
fingerling
Channa punctatus
adu 1 1
Labeo rohita
fingerling
Mastocembelus pancalus
Response manifest
at
time,
hr
96
96
96
96
115
50
60
40
51
conc.,a
ug/1
142.5
500
85 (12.7 C)
77 (23.8 C)
0.25
1.25
16.0
0.025
0.4
Type of
response
LC50
LC50
LC50
LC50
LC1009
LCI 00
LCI 00
LCI 00
LCI 00
Reference
22
24
25
25
26
26
26
26
26
Continued . . . .
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TABLE 1. CONCENTRATIONS OF CHLORDANE TOXIC TO FISH—continued
Species
Common
Tengra
Nandus
Puntl
S1ngh1
Cuchla
Carp
BluegUl
Largemouth bass
Rainbow trout
Bluegin
name
Binomial
MystusL yl ttatus
Nandus nandus
Puntla sophore
Heteropneustes
fossil is
Amphipnous cue hi a
Cyprlnus carplo
(embryos]
L. macrochlrus
Mlcropterus salmoldes
S. gairdneri
L. macrochlrus
Response manifest
at
time,
hr
60
25
18
51
45
91 3
30
87
24 3
24
cone.,8
yg/l
0.5
0.63
1.25
1.25
2
,600
200
200
,050h
21 81
Type of
response
LCI 00
LCI 00
LCI 00
LCI 00
LCI 00
slgnif. effect
on hatching
Lethal
Lethal
LC50
LC50
Reference
26
26
26
26
26
27
28
28
20
29
Continued .
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TABLE 1. CONCENTRATIONS OF CHLORDANE TOXIC TO FISH—contlnued
O-l
Species
Common
Bluegill
Bluegill'
Carp
Rainbow trout
Rainbow trout
Pike
White mullet
White mullet
name
Binomial
L. macrochirus
L. macrochirus
C. carpi o
S.. gairdneri
S. gairdneri
Esox sp.
Mug 11 curema
Muail curema
Response manifest
at
time,
hr
24
?
48
24
48
24
24
48
cone. ,a
yg/1
346J
0.1
1,160
600
10
>5
43
5
Type of
response
LC50
Increased 02
consumption
LC50
Threshold cone
LC50
Threshold cone
LC50
LC50
Reference
29
19
30
. 30
6
. 30
31
31
All chlordane concentrations were adjusted,
where possible, to 100% active ingredient.
DMedian lethal concentration.
'Salinity was 5 g/kg.
^Salinity was 25g/kg.
Hardness of 20 mg/1 CaCOg.
Hardness of 400 mg/1 CaCO-j.
^Concentration lethal to all test specimens.
LC50 calculated from concentration - % mor-
tality data of authors.
^hlordene (isomers of technical chlordane).
JPhotochlordene (photolytic degradation
product of chlordene).
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In 1962, Ludemann and Neumann (30) published extensive data on the
toxicity of insecticides to a variety of fish, invertebrates, and a species
of toad (Bufo bufo). The chlordane concentrations at which mortality just
began to occur (i.e. lethal thresholds) for carp (Cyprinus carpio), rainbow
trout (Salmo gairdneri), and pike (Esox sp.) exposed for 24 to 48 hr were
400, 600, and 5 yg/1, respectively.TRe 48-hr LC50 for carp was 1,160 yg/1.
One of the more recent reports on chlordane toxicity dealt with the insecti-
cide's lethality to several species of fish from India. Konar (26) found
chlordane to have a uniformly high toxicity, but gave LC50 values without
stating the time required for manifestation of the responses. LCI00 esti-
mates, concentrations lethal to 100% of the fish, ranged from a 115-hr value
of 0.25 yg/1 for murrel fry (Channa punctatus) to a 60-hr value of 16.0 yg/1
for murrel adults. The 24-hr LC50 value of 10 yg/1 chlordane for rainbow
trout reported by the National Technical Advisory Committee on Water Quality
Criteria (6) was one-sixtieth of the lethal threshold value reported by
Ludemann and Neumann (30) and one-fourth of that reported by Katz (23) for
rainbow trout exposed for 96 hr. Although such wide variation among labora-
tory toxicity test results reinforces the need for standardization of toxi-
city testing procedures, measurement of the actual concentrations of pesti-
cide to which the organisms were being exposed rather than reliance on the
amount added to the tanks would probably have narrowed the range in values.
Little information was found documenting the toxicity of chlordane
to marine organisms. Katz (23) exposed stickleback to chlordane at salinities
of 5 and 25 g/kg and observed the pesticide to be approximately half as toxic
at the higher salinity (96-hr LC50 of 160 yg/1) as at 5 g/kg (96-hr LC50 of
90 yg/1). In one of the few acute toxicity tests conducted with a flow-
through rather than a static system, Holden (31) reported 24- and 48-hr LC50
values for juvenile white mullet (Mugil curema) of 43 and 5.5 yg/1, respectively.
The lowest concentration of chlordane (0.1 yg/1) found to elicit a poten-
tially deleterious response (i.e. an increase in oxygen consumption) was
observed by Dowden (19) in studies of the effects of various insecticides on
bluegill. Augmented metabolic requirements of chlordane-exposed fish were
also observed by Malone and Blaylock (27) in evaluations of the toxicity of
DDT, chlordane, dieldrin, endrin, diazinon, and 0, 0, dimethyl-S-(4-oxobenzo-
triazino-3-methyl) phosphorodithioate to carp embryos. Concentrations of
chlordane below 720 yg/1 A.I. shortened incubation and stimulated embryonic
development. Twenty-three percent of the chlordane-treated embryos hatched
after 52.5 hr and 71% after 69.5 hr. None of the control embryos hatched by
52.5 hr and only 54.7% by 69.5 hr. Koch, Cutkomp and Yap (32) reported
chlordane inhibition of Mg^-ATPase activity in both mitochondrial and non-
mitochondrial preparations of bluegill brain tissue. Since ATPase converts
ATP to ADP and inorganic phosphate, inhibition of this enzyme would uncouple
oxidative phosphorylation and thereby stimulate metabolism.
Little research has been performed on behavioral responses of fish
to chlordane. Summerfelt and Lewis (33) reported that 5, 10 and 20 mg/1
concentrations of a 75% emulsifiable concentrate of chlordane repelled green
sunfish (Lepomis cyanellus), that 2 mg/1 would result in an equivocal
14
-------
response, and that 1 mg/1 would produce no response. Since these levels are
considerably greater than lethal levels, it appears that fish encountering
an acutely lethal concentration of this insecticide might be unable to detect
and avoid it.
There have been several studies of the effects of chlordane introduced
into natural watercourses. Using flow-through conditions, Cope, Gjullin and
Storm (20) introduced acetone solutions and emulsions of chlordane into
troughs and streams containing principally salmonids, caddisflies (Trich-
optera) and blackflies (Diptera-Simuliidae) in experiments designed to de-
termine whether concentrations effective in controlling the simuliids would
be deleterious to populations of salmonids and their prey. Emulsions of
1,250 yg/1 A.I. chlordane immobilized trout in 15 min and caused death within
24 hr in tests conducted in troughs. A median lethal concentration of 3,050
yg/1 was calculated from the data of Cope et al. (20) for rainbow trout
exposed to chlordane for 15 min and held for 48 hr in uncontaminated water.
In stream tests an emulsion of 1,250 yg/1 chlordane immobilized the trout in
15 min. In a later study, Mulla (34) assessed the toxicity of various insect-
icidal preparations, including an emulsifiable concentrate of chlordane, to
mosquitofish (Gambusia affinis) and bullfrogs (Rana catesbeiana), both preda-
tors of mosquitos.Applied at 0.23 kg/acre (0.51b/acre), chlordane was
moderately toxic to mosquitofish, but at 0.45 kg/acre (1.0 1b/acre), it
was highly toxic. Bullfrog mortality was judged moderate to severe at appli-
cations of 0.23 kg/acre of the emulsifiable concentrate. Dosages for insect
control are generally recommended to be less than 0.45 kg/acre.
Aquatic Invertebrates
A considerable amount of work has been performed on the toxicity of
chlordane to aquatic invertebrates. Owing perhaps to non-standardization
of test conditions and use of different response criteria, water quality,
and specimens of varying conditions, there is considerable disparity in
the test results summarized in Table 2. Toxicities ranged from a 25-hr
LCI00 of 0.33 yg/1 for backswimmers, Notonecta sp. (26), to a 96-hr lethal
threshold of 10,000 yg/1 for mussels, Dreissena polymorpha (30). Acute
sensitivities of invertebrates were generally of the same order as those
for fish.
The two most extensive studies on chlordane toxicity to invertebrates
were performed by Konar (26) and LUdemann and Neumann (30). Konar (26)
exposed nine species of aquatic insects resident in India to chlordane for
up to 168 hr and reported lethal threshold (LCO), LC50, and LCI00 concen-
trations. However, exposure times varied within the 168-hr maximum and were
not given for the LC50 values, thus limiting the Tatter's usefulness. As
noted above, Konar (26) found the backswimmer to be the most sensitive spe-
cies and the water scorpion', Nepa sp., the least sensitive (90-hr LC100 of
78.8 yg/1). LUdemann and Neumann's (30) work encompassed more taxonomic
groups than that of Konar (26). Lethal thresholds encountered with 24- to
96-hr exposure periods ranged from 1 yg/1 for the amphipod, Carinogammarus
ruesilli, to 10,000 yg/1 for V. polymorpha. The lethal thresholds for the
15
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TABLE 2. CONCENTRATIONS OF CHLORDANE TOXIC TO AQUATIC INVERTEBRATES
Response manifest
Species name
Common
Backswlmmer
Water stick
Water scorpion
Water bug
Giant water bug
Aquatic beetle
Aquatic beetle
Aquatic beetle
Dragonfly
Non-b1t1ng midge
Binomial
Notonecta sp.
Ranatra fillformis
Nepa sp.
Sphaerodema annul atum
Belostoma 1nd1ca
Hydrophilus sp.
Dytiscus sp.
Cybister
Suborder anlsoptera
Chironomus (larvae)
time,
hr
25
110
90
68
88
130
90
112
132
8
at
cone.,8
yg/1
0.33
1.0
78.8
1.25
1.25
1.58
2.0
1.25
1.0
15
Type of
response
LC100b
LCI 00
LCI 00
LCI 00
LCI 00
LC100
LCI 00
LCI 00
LCI 00
LT50C
Reference
26
26
26
26
26
26
26
26
26
36
Continued . . .
-------
TABLE 2. CONCENTRATIONS OF CHLORDANE TOXIC TO AQUATIC INVERTEBRATES—continued
Species
Common
Brine Shrimp
American oyster
Caddisfly
Amphlpod (scud)
Stonefly
Water flea
Tubiflcid worm
Mussel
name
Binomial
Artemia salina
(nauplii)
Crassostrea virginica
Hydropsyche sp.
Gammarus lacustris
Pteronarcys
californica
Simocephalus
serrulatus
Tubifex tubifex
Dreissena polymorpha
Response manifest
at
time,
hr
2-3
24
34
96
48
48
96
96
conc.,a
ug/1
10
10
l,650d
26
55
55
1,000
10,000
Type of
response
LT50
Inhibit growth
LC50
LC50
LC50
EC50e
Lethal
threshold
Lethal
threshold
Reference
37
38
20
35
6
6
30
30
Continued
-------
TABLE 2. CONCENTRATIONS OF CHLORDANE TOXIC TO AQUATIC INVERTEBRATES—continued
oo
Species
Common
Amphipod (scud)
Copepod
Isopod
Crayfish
Dlptera
(Cullcidae)
Dlptera
(Chironomidae)
name
Binomial
Carlnogammarus
rues-mi
Cyclops strenuus
Asellus aquatlcus
Cambarus afflnls
Corethra plumicornis
(larvae)
Chlronomus (larvae)
Response manifest
at
time,
hr
24
24
24
24
24
24
conc.,a
yg/1
1
1,000
50
1,000
100
>5
Type of
response
Lethal
threshold
Lethal
threshold
Lethal
threshold
Lethal
threshold
Lethal
threshold
Lethal
threshold
Reference
30
30
30
30
30
30
All chlordane concentrations were adjusted,
where possible, to 100% active Ingredient.
Lethal concentration to all specimens.
:Median lethal time.
LC50 calculated from concentration - %
mortality data of authors.
eMed1an effective concentration (EC50) is
the concentration causing immobilization
of the test specimens.
-------
amphipod and for larval midges, Chironomus sp. (i.e. 5 ug/1), were similar to
results obtained by others for species within the same taxonomic groups.
Sanders (35) determined that the 96-hr LC50 for the amphipod, Gammarus
lacustris, was 26 ug/1, while Silvey (36) calculated a median lethal time of
8 hr for Chironomus larvae exposed to 15 yg/1. The National Technical Advisory
Commission on Water Quality Criteria (6) reported that the 48-hr LC50 and
EC50 values for Stoneflies (Pteronarcys californica) and a cladoceran
(Simocephalus serrulatus) were 55 and 20 yg/1, respectively.
Very little data have been accumulated on the toxicity of chlordane
to marine invertebrates. Michael, Thompson and Abramowitz (37) determined
that 10 yg/1 would cause 50% mortality in brine shrimp nauplii (Artemia
salina) in 2-3 hr. Butler, Wilson and Rick (38) investigated the effects of
various insecticides on behavior and growth of Eastern oysters, Grassestrea
virginica, and observed inhibition of growth within 24 hr at 10 ug/1 chlor-
dane.
Field experiments of chlordane toxicity have dealt primarily with the
control of mosquitoes. Although these data will not be discussed herein,
selected references are given in the appended Bibliography (Section IX). In
an Alaskan field study of the adverse effects of insecticide applications for
blackfly eradication on native populations of fish and aquatic insects, Cope
et al. (20) found the caddisfly, Hydropsyche sp., to be the most sensitive of
three species evaluated. Fifteen minute exposure to 1,250 ug/1 chlordane
immobilized Hydropsyche sp. Calculation of an LC50 for Hydropsyche sp. using
concentration-percent mortality data given by Cope et al. (20) resulted in a
value of 1,650 ug/1. The exposure consisted of a 15-min period of insecti-
cide contact followed by 24-hr confinement in flowing freshwater.
Residues of Chlordane in Aquatic Organisms and the Environment
Following its introduction into the environment, chlordane probably
shares the same fates as many of the chlorinated hydrocarbon insecticides.
Some of the components of the technical material will vaporize and be carried
away from the point of application by thermal convection, etc. The vapory
pressure of chlordane is 1 x 10 mm Hg, more than that of DDT (1.9 x 10~ )
and endrin (2 x 10 ); hence, its volatilization might be greater than these
two compounds (15). Terrestrially, chlordane can be absorbed by organisms,
adsorbed to particulate matter (soil), or enter watercourses by dissolving in
water or sorbing to suspended particles. Chlordane is a relatively persistent
compound in the environment, a characteristic that contributes to its bio-
activity. Lichtenstein and Polivka (2) found that 12.4 to 17.8% of the
chlordane applied to turf plots remained after 12 yr in undisturbed sandy
loam soil. Heptachlor, a constituent of technical chlordane, had disappeared
completely after 9 yr in silty clay loam soil. In comparison, Miami silt
loam and muck soils treated with 10 and 100 Ib/acre DDT retained approxi-
mately 22 and 33% of the insecticide after 3.5 yr (39). Thus, chlordane is
persistent relative to other organochlorine pesticides in soil.
19
-------
Stability to chemical, physical, and biological degradation is one of
the prerequisites for a chemical to be available for uptake by organisms
(bioaccumulation) and accumulated and transferred up food chains (biomagni-
fication). There are several reports in the literature indicating that
chlordane is accumulated, but no studies were found documenting biomagni-
fication of this pesticide. Godsil and Johnson (40) found that DDT, chlor-
dane and endrin, when applied seasonally during the summer growing season,
would not accumulate in the aquatic food chain over successive years, but
rather would decrease during winter to the limits of detection in both water
and the biota. During the growing season, however, when the water contained
up to 0.100 ug/1 chlordane, algae (Cladophora sp.) were found to contain up
to 50 ppb chlordane; vascular plants (Hyriophyllum sp. and Potamogeton sp.),
to 67 ppb; chubs (90% Siphateles bicolorandTOTT. gila), to 24 ppb; and
clams (Gonidea sp.), to 12,0 ppb.Four different groups of largemouth bass
and two groups of clams were also held for varying periods in cages in the
same stream in which the natural populations of plants and animals were
sampled. Largemouth bass accumulated from 8 to 43 ppb chlordane in less than
120 days, whereas accumulation by clams was 2 to 25 ppb (40). In a far
more extensive pesticide monitoring program, Henderson, Johnson and Inglis
(41) collected and analyzed 62 species of fish from the Great Lakes and major
U.S. river basins for nine pesticides and their metabolites. Roughly 128 of
the 587 composites of fish sampled (21.8%) contained detectable residues of
chlordane. Whole body contents ranged to 7.3 ppm. The Gulf Coast fish con-
tained the highest incidence of chlordane (45.8%) and the Columbia River
system the least (1.7%). Green et al. (42), in a survey of 109 sites in U.S.
rivers, stated that aqueous concentrations of chlordane in situ were only
0.1 yg/1.
Although the oceans can be regarded as an ultimate depository for a
proportion of the chlordane applied on the mainland, a recent study by Duke
and Wilson (43) on the contents of insecticides in 29 species of marine fish
from the West Coast of the United States revealed no detectable residues of
chlordane. While this finding might imply that chlordane's stability and
susceptibility to biomagnification are less than those of DDT and its meta-
bolites, for which residues up to 1,026 ppb were measured by Duke and Wilson
(43), analytical methods for DDT and its metabolites are generally much more
sensitive than those for chlordane and may, in fact, obscure chlordane on a
chromatogram (personal communication, L. Mueller, EPA, Environmental Research
Laboratory—Duluth). Hence, results of pesticide residue surveys in situ
may not necessarily reveal the extent of chlordane biomagnification in natu-
ral populations of aquatic organisms.
20
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SECTION V
MATERIALS AND METHODS
ACUTE TOXICITY TESTS
Test Species
Fish— The acute lethal toxicity of technical chlordane was examined using
Brook trout, Salve! inus fontinalis (Mitchill), bluegill, Lepomis macrochirus
Rafinesque, and fathead minnow. Yearling brook trout were obtained from the
California State Department of Fish and Game, held for 1 year, and tested as
adults. Bluegill were obtained as juveniles from a local commercial dealer,
while fathead minnow were reared in the laboratory from stock that had been
originally obtained from the U.S. Environmental Protection Agency's Environ-
mental Research Laboratory (ERL-D) at Duluth, Minnesota. The ages and sizes
of the fish used for testing are given in Table 3.
Invertebrates— The crustacean, Daphnia magna Straus (Branchiopoda, Cladocera),
was obtained from ERL-D and cultured in both continuous-flow and static sys-
tems. The non-biting midge, Chironomus No. 51 (Diptera, Chironomidae), was
obtained from the University of California's Department of Entomology at
Riverside, and the amphipod or "scud", Hyallela azteca (Saussure), was col-
lected from small, constant temperature (16°C) streams immediately adjacent
to the California Department of Fish and Game's Fillmore Hatchery.
Acclimation and Toxicity Testing Conditions
All acute toxicity tests were conducted in accordance with methods
recommended by The Committee on Methods for Toxicity Tests with Aquatic
Organisms (44) and Sprague (45).
The fish were acclimated to toxicity test conditions for at least 2
months under controlled conditions. Brook trout and bluegill were fed a dry
pelleted ration (Moore-Clark Co., Salt Lake City, Utah), the former at 2% of
their body weight per day and the latter ad_ libitum twice daily. Fathead
minnow fry were fed a mixture of 50% brine shrimp nauplii, 25% dry trout
starter (Moore-Clark), and 25% "TetraMin" (Tetra-Werke, West Germany), ad^
libitum twice daily. Three days prior to conducting an acute toxicity test,
10 fathead minnow, 10 bluegill, or 5 brook trout were randomly distributed
species has not been described, but has been classified in the interim
as No. 51 by the University.
21
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TABLE 3. CHARACTERISTICS OF FISH EXPOSED TO TECHNICAL CHLORDANE IN
ACUTE TOXICITY TESTS
to
N)
Approximate
age at testing,
Species months
Brook trout
Test 1 24
Test 2 24
Fathead minnow 3
BlueglH 3
Developmental
stage
Aa
A
Jc
J
Density 1n
test chamber,
g f1sh/l
32.8
43.0
o.n
0.41
Total
length
mm
233b
+14
248
+15
26.2
+4.5
50.8
+6.4
Wet body
weight
g
131
+26
172
+36
0.18
+0.10
1.85
+0.75
aAdult.
bMean +_1 standard deviation.
C0uvenile.
-------
into each of 12 randomly positioned test chambers. The orders of tank place-
ment were established with a random numbers table. The large size of the
brook trout necessitated use of smaller sample numbers. Unfortunately, much
smaller trout or larger test chambers, though highly desirable, were unavail-
able at the time these tests had to be carried out. Test chambers were
constructed of glass and silicone rubber cement (Dow-Corning). Those uti-
lized for the brook trout and fathead minnow tests measured 30.5 x 30.5 x
30.5 cm, with water depths of 21.5 cm (20 1) and 17.7 cm (16.5 1), respec-
tively. Bluegill were exposed in 91.4 x 30.5 x 30.5 cm glass chambers con-
taining 4.25 1 of water at a depth of 15.2 cm. For all three species, 2-1
proportional diluters (46) supplied each of the 12 test chambers, which
comprised five toxicant concentrations and a control in duplicate. Each
diluter supplied sufficient water to replace 10 tank volumes per day, a rate
which insured 90% molecular replacement in 6 hr. Toxicant concentrations
were successively diluted by a factor of 0.75. A syringe dosing device
delivered microtiter amounts of the toxicant at each diluter cycle. During
the 72-hr acclimation to test conditions and the 96-hr and 192-hr toxicant
exposure periods, the fish were not fed. A photoperiod of 16 hr was employed
in all experiments. Light intensity from fluorescent lamps (Sylvania "Gro-
Lux" and Durotest "Optima") averaged 1010 lux (lu). Black plastic curtains
were used to shield the fish from disturbance. Water temperatures in tests
with bluegill and fathead minnow were thermostatically regulated at 25°C in
air-conditioned rooms. Those with brook trout were similarly controlled at
15°C. Although water flow into the tanks maintained dissolved oxygen con-
centrations above 70% of air saturation in most tests, artificial aeration
was required in those using trout to meet this requirement.
Dead fish were measured for total length to the nearest millimeter
and, after excess moisture had been removed with toweling, for wet body
weight to the nearest gram or milligram, depending on size. All fish were
measured from the six treatments constituting one replicate. The data were
later pooled when no size differences between treatments were detected.
Length-weight measurements were not taken prior to toxicity testing since it
was believed that the stresses associated with handling and anesthesia (47,
48, 49) would have a greater influence on the LC50 than the changes in body
weights during the course of the test.
Daphnia magna were cultured with both static and flow-through condi-
tions at room temperature (20° to 21°C). Those held in the static system
were fed once daily and those in the flow-through system twice daily. The
ration consisted of a blended and sieved (100 mesh) mixture of dried baker's
yeast (4.5%), pelleted fish food (91%) and alfalfa (4.5%) in water. Second
to third instars were used for testing, and they were not fed during toxicant
exposure.
The amphipod, FL azteca. was cultured in a flow-through system at 16°C
and fed pre-soaked aspen leaves, supplemented with dry pelleted fish food.
Live Myriophyllum sp. was also introduced as a possible food supply and
habitat^Juveniles (~ 5 mm in total length) were used for acute toxicity
testing and were not fed during the test.
23
-------
The sensitivity of Chironomus No. 51 to technical chlordane was not
evaluated successfully in an acute toxicity test because a satisfactory
means of testing was not found. Use of a substrate of fine sand, the pre-
ferred habitat of this species in the laboratory, would not permit temporal
examination of the status of the test specimens without causing considerable
disturbance, whereas preliminary trials indicated that confinement of the
larvae in egg incubation cups resulted in a random, anomalous mortality.
Cladocerans and amphipods used for acute toxicity testing were acclimated
to test conditions, i.e., photoperiod and temperature, but not to the specific
test apparatus prior to introduction into the chambers. Shortly before
chlordane exposure, 10 specimens were randomly distributed into each of the
randomly positioned test chambers. The test chambers consisted of glass
cylinders, 6.5 cm inside diameter (i.d.) and 7.5 cm long, suspended in 30.5 x
30.5 x 30.5 cm glass chambers. Nylon screen ("Nitex", 500 y openings) was
attached to one end of the cylinder with silicone rubber cement (Dow-Corning)
to permit circulation and retain the specimens. Each of the large chambers
contained two cylinders and was supplied with appropriate test concentra-
tions from a 2-1 proportional diluter (46). Chlordane solutions were deliv-
ered in microliter amounts to the diluter's mixing (M-l) cell with the syringe
dosing device described by Mount and Brungs (46). Toxicant concentrations
were successively diluted by 25%. The tests with D. magna lasted 96 hr. The
test utilizing H. azteca was extended to 168 hr since mortalities were minimal
within 96 hr, even though the highest calculated chlordane concentration
tested approximated the solubility limit.
The response criterion of immobilization was satisfied when the speci-
mens lay motionless on the bottom and did not move when gently prodded.
Water Quality
The diluent water for all tests was supplied from local wells and was
unchlorinated except for treatment of storage reservoirs for algal control
1 day per week. Total residual chlorine was not detected upon periodic
measurement (leucocrystal violet method of APHA [21]), even on the day of
chlorination (Friday). As a precautionary measure, however, activated carbon
filters were installed on the line supplying the £. magna, Chironomus No.
51, bluegill, and fathead minnow acute and chronic tests. Due to high flow
requirements (76,000 I/day) of several brook trout chronics being conducted
concurrently, the associated high cost of dechlorination equipment, and the
belief that there was a low probability of a chlorine toxicity problem, no
activated charcoal filters were installed on this line which supplied all
trout as well as the amphipod tests. As will be seen, mortalities believed
due to residual chlorine were observed near the end of this research project
in approximately 20- to 40-day trout alevins, but not in older or younger
trout or in the other species.
Seven water quality variables were monitored routinely during each
test: water temperature, dissolved oxygen concentration, pH, total alkalinity,
acidity, total hardness, and specific conductance. Except for water tempera-
ture, which was recorded continuously, water quality in the test chambers was
24
-------
measured 24 hr prior to and at least once during chlordane addition for com-
parison of the effects of the toxicant's presence on water quality, as a check
on the water's suitability for uncompromised organism survival, and for esti-
mation of water quality variation. All variables were determined with standard
methods (21, 50). Except for rare instances, measurements were made on samples
collected less than 4 to 6 hr earlier.
A number of ions were also determined by a commercial laboratory to gain
a more complete description of the water's composition. Calcium, magnesium,
potassium, sodium, chloride, and sulfate ions were determined every 4 months
over 1 yr, ammonia was measured biannual ly and the other compounds once
(Appendix Table 2).
CHRONIC TOXICITY TESTS
Fathead Minnows
The design, apparatus and conditions employed for the chronic toxicity
tests using fathead minnows were developed by the U.S. Environmental Protec-
tion Agency (51). The basic apparatus consisted of a 2-1 proportional dilu-
ter which supplied successively diluted (by a factor of 0.5) concentrations
of technical chlordane to twelve 42.5-1 (91.4 x 30.5 x 30.5 cm) glass tanks,
comprising five toxicant concentrations and a control in duplicate. Daily
flow through each chamber averaged six tank volumes, assuring 90% molecular
replacement in approximately 9 hr. After spawning commenced, two glass
chambers (28.5 x 14 x 15 cm) were placed into one end of each adult tank for
rearing the progeny. All three chambers were supplied separately with test
water, although effluent from the fry chambers passed directly through
screening into the tank containing the adults.
The test was conducted with a photoperiod regulated to produce gonadal
recrudescence and senescence at a cycle simulating that existing at Evansville,
Indiana. Sunlight was simulated with Durotest "Optima" and Sylvania "Glo-
Lux" fluorescent bulbs and the intensity averaged 1010 lu.
The water temperature was 21°C upon initiation of the chronic test,
but was raised within the first 8 weeks to 25°C and maintained at that
temperature thereafter.
At the beginning of the chronic toxicity test, 5-day-old fathead minnow
fry were randomly distributed into one fry chamber (50 specimens each) in
each of the 12 adult chambers. The fish were initially fed Oregon Moist
Pellet starter mash (Moore-Clark) and "TetraMin" tablets crushed to a fine
powder. Since growth and survival were poor, the diet was replaced after 2
months with one of frozen brine shrimp nauplii. As the fish grew, they were
fed increasing proportions of frozen adult brine shrimp and dry trout pellet
(0.047 mm dia.).
Reduction in the density of f0-generation test specimens, "thinning",
was not undertaken after the recommended 60-days* toxicant exposure because
of appreciable mortality in all test chambers. Rather, excess fish were
25
-------
removed after 5.5-months1 chlordane exposure, just prior to anticipated
spawning. Concurrently, five spawning substrates, consisting of halved 10 cm
i.d., red-clay channel pipe were placed into each adult chamber. Spawning
commenced within 24 hr, indicating that the substrates could have been intro-
duced earlier.
From each spawning, all embryos were counted and one to several groups
of 50 eggs incubated to determine hatching success. When there were fewer
than 50 embryos per spawn or when embryos were spawned on weekends, they
were only counted. Incubation cups consisted of polypropylene pipe, 7 cm
long and 5 cm i.d., covered at one end with "Nitex" screen (500 y openings).
The cups were oscillated continuously with a rocker-arm assembly (52). At
hatching, the numbers of normal, abnormal (i.e. having vertebral abnormali-
ties or otherwise abnormal morphology or behavior), and dead fry were counted.
Each rearing chamber was stocked with 50 fry from concurrent hatches, and the
fj-generation progeny reared. After 30 days' growth, fry were captured and
photographed using the method of McKim and Benoit (53) for measurement of
total length. After 60 days all fry were sacrificed and measured to the
nearest millimeter for total length and weighed to the nearest milligram
after removal of external moisture with toweling. In the first photographic
measurement, 20.3 x 25.4 cm black and white prints were made. Thereafter,
slides were made of the negatives and projected onto a screen for measure-
ment of lengths. This was believed to have enhanced measurement accuracy,
owing to the larger image, and was less costly.
The chronic toxicity test utilizing fathead minnows was terminated
after all adults had completed spawning and all fry had been reared for
60 days. Adults were weighed, measured, sexed, and examined for general
condition.
Bluegill
The partial chronic toxicity test utilizing bluegill was conducted
with a method recommended by the EPA (54). Since the experiment was not
begun with embryos or fry, but with yearlings, the test constituted a partial
rather than full chronic because it did not encompass one complete genera-
tion. The experimental apparatus consisted of a 2-1 proportional diluter and
12, randomly positioned, 91.4 x 61 x 38 cm tanks containing 178 1 of water at
a depth of 32 cm. The tanks were illuminated at an average intensity of 1010
lu by two fluorescent lamps (Durotest "Optima" and Sylvania "Gro-Lux").
Photoperfod was regulated to simulate that existing at Evansville, Indiana.
The proportional diluter delivered 10 tank volumes per day to each chamber,
assuring 90% molecular replacement in about 5 hr.
At the beginning of the test (5 December 1972), juvenile bluegill,
obtained from a commercial fish breeder, were anesthetized with ethyl m-
amlnobenzoate methanesulfonlc add salt (tricaine methanesulfonate), weighed,
measured for total length, and 20 specimens randomly distributed into each of
the 12 chambers. They were fed twice dally ad libitum a dry pelleted ration
(Moore-Clark). After 3, 5, and 9.5 months' toxicant exposure, all surviving
fish were captured, anesthetized, weighed, and measured for length to
26
-------
determine relative growth.
At 5 months the density of fish in each tank was reduced to three males
and seven females in anticipation of spawning. Those which appeared to be
sexually immature were discarded.
TU .Atter,6 moths' exposure (6 June 1973) bluegill commenced spawning.
The i-nitial spawning occurred 1 month after two 30.5 x 30.5 cm gravel-cement
spawning substrates had been placed into each of the 12 chambers. Each
substrate had a 24-cm oval depression 4 cm in depth. Generally, fish spawned
in the depression, but eggs also tended to be deposited in adjacent areas of
the tank, due in part to turbulence produced by the spawning activity. All
substrates were checked daily for spawns. Embryos were brushed from the
substrate and a number taken for determination of percentage hatch and for
the growth and survival studies. The remainder were preserved in 5% formalin
for later estimation of spawn size. From each spawn, groups of 100 embryos
were incubated in a manner similar to that for fathead minnows, except that
10-1 glass rearing chambers (30.5 x 30.5 x 15 cm), which were situated down-
stream from the 178-1 tanks and received test water directly from them (six
tank turnovers per day), were used for incubating and rearing the progeny.
Substantial embryo mortality consistently occurred as a result of the fungus,
Saprolegnia sp., spreading from dead to contiguous living eggs. Although
efforts were directed toward obtaining only live embryos for incubation, the
substantial amounts of organic debris associated with the adhesive embryos
made it difficult to obtain only live embryos or separate dead embryos from
within masses of living ones. Treatment of the embryos with 4 mg/1 zinc-free
malachite green, the level prescribed by Smith (55) for treating flagfish
(Jordanella floridae) embryos and found by us to be of some value for treat-
ing fathead minnows, was abandoned after initial 5 min baths caused substan-
tial mortality.
Upon hatching, the proportions of normal and abnormal fry were deter-
mined and all normal fry reared. Abnormal fry were segregated visually on
the basis of physical (e.g. vertebral) defects and erratic behavior. Fry
were initially fed blended, cooked chicken egg yolk and "green" water (com-
posed of unicellular chlorophyte algae, protozoans, and copepods) until they
became large enough to consume frozen brine shrimp nauplii and "Tetra-Min"
tablets crushed to a fine powder. Fry larger than approximately 15 mm in
length were offered frozen adult brine shrimp. Acceptance of the dry diet
may have been limited. At 30 and 60 days, fry were transferred into a 30.5 x
30.5 cm glass chamber filled with 1.3 cm water and photographed (53). Total
lengths of the fry were determined with reference to a metered grid from
black and white negatives projected from slides. After 90 days, surviving
fry were measured manually for total length and for wet body weight. After
the adults had completed spawning and the fry reared for 90 days, the chronic
toxicity test was terminated. Adults were weighed, measured for total length,
sexed, and examined for general condition.
27
-------
Brook Trout
The partial chronic toxicity test using yearling brook trout was begun
on 29 March 1973 using procedures and conditions recommended by the U.S.
Environmental Protection Agency (56). Just prior to their introduction, the
brook trout were anesthetized, measured for total length and weighed. Twelve
fish were placed randomly into each of the randomly positioned chambers.
The toxicity testing apparatus consisted of a 4-1 proportional diluter
and twelve 91.4 x 61 x 38.1 cm glass chambers containing 178 1 of water at a
depth of 32 cm. The diluter delivered approximately nine tank volumes per
day (1602 1), assuring 90% molecular replacement in 5.5 hr. Technical chlor-
dane concentrations were successively diluted by a factor of 0.5. Each tank
was covered with screening to retain the fish, and pairs of tanks were illum-
inated with fluorescent lamps (Durotest "Optima" and Sylvania "Gro-Lux") at
an intensity of 1010 lu. The photoperiod simulated that existing at Evans-
vine, Indiana, and the temperature followed the cycle recommended by EPA
(56). When the numbers of trout in each tank were reduced in anticipation of
spawning, black plastic was used to cover the sides and tops of the tanks in
an effort to minimize antagonistic behavior between males in adjacent tanks
and provide a more secluded environment for spawning.
Shortly after the test was begun, antagonistic behavior between the
fish was observed. Hierarchies were eventually established, but not without
considerable fighting. Consequently, up to two fish in each tank developed
Saprolegm'a sp. infestations. Initially, all tanks were treated for 1 min
with 67 mg/1 zinc-free malachite green and the fungoused areas of the fish
painted with a 200 mg/1 solution of the compound (57). Both methods were
unsuccessful, and the fish perished. After testing different concentrations
of malachite green, a level of 0.2 mg/1, employed as a "flush" treatment, was
found to be successful in preventing outbreaks of the fungus, but not in
arresting an advanced infestation. These treatments were employed once daily
for 2 weeks after the fish were handled (i.e. after measurement of growth at
3, 6.5, and 12 months) and during spawning. Several fish also developed what
appeared to be bacterial hemorrhagic septicemia subsequent to their initial
introduction and after assessment of growth at 3 months. This was success-
fully treated by incorporating oxytetracycline ("TM-50", Pfizer) into the
feed to give a concentration of 0.44% active ingredient or 75 mg A.I./kg/
fish/day. Use of oxytetracycline at this level cured some fish and pre-
vented further outbreaks of the disease.
During the test, brook trout were fed a dry pelleted ration (Moore-
Clark) at a rate of 2% of their body weight per day. At 3, 6.5 and 12 months,
the trout were measured for length and weighed to determine relative growth.
After 6.5 months, the numbers of fish residing in each tank were reduced to
three males and four females in anticipation of spawning. Since only two of
the fish held in 5.8 yg/1 chlordane remained, excess control specimens were
transferred to one of the 5.8 yg/1 tanks. Upon reduction in specimen density,
two glass spawning chambers, 30.5 x 25.4 x 10.2 cm, were placed into each
tank. The chambers, described by Benoit (58), were designed to simulate a
redd.
28
-------
Brook trout began spawning on 28 December 1974, 8 months after intro-
duction and 1.5 months after "thinning". Each day the number of spawns
and embryos per spawn were recorded, and embryos from selected spawns placed
into an incubation apparatus (52) for determination of hatching success or
viability (development of a neural keel after 12 days). Black plastic was
used to shield the developing embryos from light. Viability determinations
were usually made on every spawn totaling 20 embryos or more. Determinations
of hatching success, which utilized 50 embryos collected from a single spawn-
ing, were spread essentially equally throughout the spawning period. Up to
eight such determinations were made from the spawns from each of the 12 adult
tanks (16 determinations per treatment). Data collected in the hatch study
included times to hatching of 50% of the alevins, percentages of normal,
abnormal and dead alevins, and total lengths and weights of the alevins.
Lengths and weights were determined only for the alevins discarded at hatch-
ing.
To assess the effects of chlordane on growth and survival of the progeny,
groups of 25 alevins each were reared for 90 days in each of the treatments.
Up to four groups of alevins were ultimately used per concentration. These
studies were conducted in 37.5 x 18 x 13 cm glass chambers (10 cm depth)
which were separated from the adult tanks, received water directly from the
diluter, and were covered with screening to retain the fish.
Fry were fed Oregon Moist Pellet trout starter (Moore-Clark) until
they were old enough to consume adult brine shrimp and dry pellets. After
30 days' growth, fry were measured for total length using the photographic
method (53). At 90 days, fry were measured, weighed and killed.
When the adults from all chambers had not spawned for 2 weeks, they
were killed, weighed, measured for total length, and examined for general
condition.
Hyallela azteca
The chronic toxicity test utilizing H. azteca was conducted according to
a procedure of EPA (59) and a system consTsting of a 2-1 proportional diluter
and twelve 17.5 x 20.5 x 25 cm glass tanks. Each tank contained 8.3 1 of
test solution at a depth of 23 cm and was immersed in a water bath to mini-
mize fluctuations in water temperature. The diluter replaced four tank
volumes (i.e., 33 1) in 24 hr, a rate equivalent to 90% molecular replacement
in approximately 15 hr.
On 26 March 1974, 25 newly hatched H. azteca were introduced into each
of the twelve chambers comprising five chlordane concentrations and a control
in duplicate. The photoperiod was held at a constant 16-hr light and the
water temperature at 17°C. Aspen leaves (Populus sp.), soaked in water for
30 to 60 days prior to feeding, and live Myriophyllum sp. were introduced as
food and habitat. Small (2 mm) pellets of fish food (Moore-Clark) were
introduced periodically to supplement their diet. The value of the plant and
fish pellet as food for this amphipod species was unknown, but both were
included because specimens in stock cultures appeared to consume them. The
29
-------
aspen leaves were definitely of dietary importance.
Amphipods were reared under the above conditions for the 65 days of
toxicant exposure. Due to time constraints, the test could not be extended
through reproduction, although copulating pairs and ovigerous females were
observed at termination. At the end of the test, the contents of the test
chambers were successively passed through 8, 24, and 100 mesh stainless steel
screens (W.S. Tyler Co.) to isolate the amphipods. They were then placed on
tissue paper to remove excess moisture and individually weighed on an analy-
tical balance. All specimens from each chamber were then dried to constant
weight at 50°C, and the dried specimens analyzed for chlordane residues.
Daphnia Magna
The chronic toxicity test utilizing Daphnia magna was conducted accord-
ing to a procedure recommended by EPA (607- A 500-ml proportional diluter
delivered technical chlordane to a duplicated series of five concentrations
and a control. Each glass test chamber measured 28 x 13.5 x 15 cm and con-
tained 5.7 1 of test solution. The diluter delivered 3 1 to each tank daily,
equivalent to replacement of 0.5 tank volumes per day or 90% molecular re-
placement in approximately 100 hr. Higher flows are believed deleterious to
this species (personal communication, K.E. Biesinger, EPA, ERL-D). The
diluent was tap water that had been aged at least 2 days in sunlight. Aged
rather than ambient tap water was instituted to insure removal of any residual
chlorine, which is known to produce effects on D. magna at levels as low as 3
yg/1 (61).
The 4-week test was begun by introducing 10 first instars, obtained
from adults reared in a continuous-flow system, into each of the 12 chambers.
The photoperiod was a constant 16-hr light and the water temperature approxi-
mately 21°C. The cladocerans were fed daily the blended and sieved mixture
of dried yeast, pelleted fish food, and dried alfalfa grass described earlier.
Numbers of surviving f -generation daphnids and f,-generation progeny were
counted weekly and the°progeny removed. At termination, the progeny produced
in the fourth week were removed, composited by treatment, dried to constant
weight at 50°C, weighed on an analytical balance, and analyzed for chlordane
content.
Chironomus No. 51
The conduct of the chronic toxicity test of the midge, Chironomus No.
51, was similar to that recommended for £. plumosus (62). Chlordane was
mixed and apportioned in a 2-1 proportional diluter operating at a rate suf-
ficient to replace 11.4 tank volumes per day (90% molecular replacement in
approximately 5 hr) in 38 x 13 x 18 cm glass chambers containing 4.2 1 of
water. Each container was covered with screen to contain the adults upon
their emergence. The toxicity test was conducted at a water temperature of
25°C and a photoperiod of 16 hr light. The dimming device to simulate dawn
and dusk and induce copulation was not used for several reasons. First, it
was believed that evaluation of toxicant effects on reproduction had a low
probability of success because the helical arrangement of the embryos in
30
-------
the skeins prevented their segregation and accurate enumeration. Secondly,
absence of dawn and dusk periods was intended to temporarily delay oviposi-
tion, which was known to occur regardless of the occurrence of copulation,
and allow harvest of the gravid adults. The latter was apparently successful
since only one to two spawnings were observed during the course of testing.
At the beginning of the test, newly hatched larvae were randomly distributed
into-each of the test chambers. They were fed "TetraMin" flakes twice weekly.
Adults were captured, sexed, and composited for total dry weight measurement.
After determination of dry weight by drying to contant weight at 50°C, the
adults were analyzed for chlordane residues.
Water Quality Analysis
In each chronic toxicity test, six water quality variables, namely
dissolved oxygen concentration, pH, total alkalinity, total hardness, and
specific conductance, were monitored in the test chambers every week. Repli-
cates were measured on alternate weeks. Water temperatures were continuously
recorded and a representative reading taken daily. Measurement of acidity
was instituted toward the middle of the project. In the first chronic toxic-
ity tests (fathead minnow and bluegill), measurements were made in each of
the six tanks comprising a replicate. Since variation in water quality
between treatments was small and was not altered by the presence of chlordane,
the carrier, or Triton X-100, water quality analyses in the trout, cladoceran,
amphipod, and chironomid tests were confined to the control, mid-range, and
high concentrations. Methods of analysis were the same as those described
for the acute toxicity tests.
ANALYSIS OF TECHNICAL CHLORDANE
Stock solutions of technical chlordane were prepared by dissolving
the insecticide in double distilled, pesticide-free acetone containing a
small amount of the nonionic surfactant, Triton X-100 (Rohm and Haas).
The surfactant was intended to enhance the rate of chlordane dissolution
in the proportional diluter and decrease the difference between desired
and measured insecticide concentrations. The expected concentrations of
Triton X-100 were half the nominal level of technical chlordane for both
acute and chronic experiments. Thus, for example, in the chronic tests
with the three fish species, where desired chlordane concentrations were
0.625, 1.25, 2.5, 5.0 and 10.0 yg/1, the nominal concentrations of the
surfactant were 0.3, 0.6, 1.3, 2.5 and 5.0 yg/1, respectively. Although
solvent controls were not employed in the chronic tests, the acute and
chronic effects of Triton X-100 on brook trout and fathead minnow have been
examined in a separate program (63), and concentrations having no chronic
effect were uniformly above 100 yg/1.
All water and invertebrate tissue samples were analyzed for technical
chlordane with a gas chromatograph equipped with a 5JNi electron capture
detector (Model 990, Perkin-Elmer Corp.). The 183 cm long, 0.64 cm o.d.
glass column was packed with 2% SE-30 on 100 - 120 mesh "Gas Chrom Q".
The carrier gas was nitrogen, and the flow rate was 60 ml/min. Oven tem-
peratures varied depending upon the type of sample being analyzed.
31
-------
For measurement of technical chlordane in water, 10.0 to 20.0 ml water
samples were extracted once for 1 min with 2.0 to 5.0 ml portions of pesti-
cide-grade n-hexane. The larger sample volumes were applied to determinations
made in the chronic toxicity tests, whereas smaller sample volumes sufficed
for measurements made in acute toxicity tests. One to 10 microliters of
sample extract were injected into the chromatograph. The chromatograms
were measured for peak area with a planimeter and compared to technical
chlordane standards. Separation of the chlordane constituents was performed
with isothermal conditions (200°C) and detector, manifold, and injector
temperatures of 240°C.
The accuracy and reproducibility of the method were checked by spiking
technical chlordane into laboratory water. Samples containing technical
chlordane concentrations of 10 ug/1 were analyzed with a recovery of 100.2
j^5.2%, while concentrations as low as 0.5 yg/1 were recovered to the extent
of 87.2 +_ 11.035. The coefficients of variation for the 0.5 and 10.0 yg/1
concentrations were 12.6 and 5.2%, respectively.
Analyses of the contents of the various technical chlordane constituents
in the three fish species were considered unreliable due to several technical
errors which were not identified and corrected in time to have the analyses
repeated. These problems were corrected prior to analyses of technical
chlordane residues in the three invertebrate species.
Whole body extracts of dried samples of the invertebrates were obtained
by grinding the specimens in a tissue grinder with pesticide residue-free
petroleum ether (30° to 75°C). One to 10 microliter volumes of the extract,
which was used without further manipulation, were separated on the gas chroma-
tograph using a 160° to 240°C temperature program (6°C/min) and the operating
conditions described for chlordane analysis in water. No major interferences
were detected in the chromatograms; heptachlor, the chlordenes, cis-chlordane,
trans-chlordane, cis-nonachlor, and trans-nonachlor were selectedTfor quanti-
tation.
f
STATISTICAL ANALYSIS
Acute Toxicity Tests
Concentration-percent mortality data were analyzed with logarithmic-
probability (log-probit) methods using either the manual procedure of Litch-
field and Wilcoxon (64) or the computer program of Dixon (65). Computer
processing was accomplished using IBM 360/75 hardware. The log-probit method
was selected because it is a more objective approach than the graphical
interpolation method, offers a test of the regression line's goodness-of-fit,
and provides the statistics necessary for calculating 95% confidence limits
for median lethal concentrations (LC50) and for comparing differences between
two LC50 values.
For homogeneous data, upper and lower 95% confidence limits for LC50
value were calculated as (LCSOHfl and LC50/f, respectively, where f is the
antilogarithm of 1.96 6 (N'/2) ' , 6 is the standard deviation of the
32
-------
logarithm of the population tolerance frequency distribution, and N1 is the
number of test animals expected to have perished within the percent mortality
interval of 16 to 84% (64). An equivalent equation is +_ 1.96 times the
standard error of the log LC50 (66). For heterogeneous data, i.e. where
chi-square analysis of the fitted line indicated lack of goQdness-ofjfit,
the following equation was used: f = (Student's t-value) (a) (x /n)
(N'/2~ ' , where n is the number of concentrations used in calculating the
LC50 (66). The logarithms of the median lethal concentrations were plotted
against the logarithms of the exposure times to give toxicity curves (45)
for the fish species and JD. magna.
The accuracy of the LC50 estimates and their 95% confidence limits,
generated by the Litchfield and Wilcoxon (64) and computer program (65)
methods, were compared with similar calculations made by eight other aquatic
toxicology laboratories using standard concentration-percent response data
(Appendix Table 3) supplied by The Committee on Methods for Toxicity Tests
with Aquatic Organisms. Our LC50 estimates were in agreement with the
average LC50 computed by the other laboratories (Appendix Table 4) using
both methods of analysis, but the 95% confidence limits were usually nar-
rower with the computer method than with that of Litchfield and Wilcoxon
(64).
Median lethal times (LT50) for measured toxicant concentrations were
calculated in some cases. The LT50 is the time required for 50% of the
test specimens to die in a given concentration. Data were analyzed in
the same manner as for the calculation of LC50 values and were plotted
on the same toxicity curve, with the exception that 95% confidence limits
were determined for the independent variable, time, rather than the LC50.
Control mortality occurred in less than 5% of the toxicity tests and
was less than 10% in all cases. Median response estimates were corrected
for any control mortality with the computer program method.
Several statistical comparisons were made to determine the significance
of differences between the 96-hr LC50 values of the different species using
data generated by probit analysis. The equation, Student's t = (Oj = f^)
(6,2/1 /2 + 622/N£ /2)"1/2, was used to test significance. The symbol 0
denotes an LC50 and subscripts pertain to the particular LC50 values being
compared. The degrees of freedom were n-, + n~ - 2, where n is the total
number of test specimens employed to derive an LC50 estimate.
Chronic Toxicity Tests
Analysis of variance with a one-way design was used exclusively in
evaluating the significance of differences between treatments. Dunnett's
test (67) was used to determine whether controls were different from each of
the other treatments. Log-probit analysis was used to determine median
emergence times of adult chironomids in the chronic tests of that species.
33
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SECTION VI
RESULTS
ACUTE TOXICITY TESTS
Water Quality and Toxicant Concentrations
Water quality during the various acute toxicity tests is summarized
in Table 4. Water temperatures were 25°C in tests of fathead minnow and
bluegill, 15°C in the tests of brook trout and H. azteca. and 21°C in that
of D. magna. Dissolved oxygen concentrations were greater than 70% in all
toxTcity tests except the second one employing brook trout where it was 62%
of air saturation. The diluent water was moderately alkaline and of inter-
mediate hardness.
Concentrations of technical chlordane were determined from two to
five times during each test, depending upon exposure time, and are summarized
in Table 5.
Toxicity
The order of decreasing species sensitivity to acutely lethal concen-
trations of technical chlordane was D. magna, fathead minnow, brook trout,
bluegill, and H. azteca. Median effective concentrations ranged from 96-hr
values of 28.4 and 35.2 yg/1 for J). magna to a 168-hr value of 97.1 yg/1 for
H. azteca. Too few H. azteca had perished at 96 hr to permit an EC50 esti-
mate for this period~bf exposure (Appendix Table 5). The 96-hr LCBO's were
36.9 yg/1 for fathead minnow, 45 yg/1 for brook trout, and 59 yg/1 for blue-
gill (Appendix Tables 6, 7, and 8).
Comparisons were made to determine the significance of differences
between species in terms of their respective 96-hr EC50 and LC50 values.
As shown in Appendix Table 9, the only statistically significant differences
were between £. magna and bluegill and brook trout (p < 0.001). None of the
fish species were significantly different 1n sensitivity.
Chlordane exposures were sufficiently long (up to 192 hr) in the acute
toxicity tests to allow partial delineation of toxicity curves (F1g. 2).
Linear curves described the toxicity of chlordane to brook trout, bluegill,
and possibly to D. magna, whereas a rectangular hyperbola characterized its
toxicity to fatheWTntrufows. The 192-hr LC50 of 32 yg/1 may be considered
an estimate of the median lethal threshold of technical chlordane for fat-
head minnow—the concentration at which acute lethality to 50% of the test
specimens ceases.
34
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TABLE 4. WATER QUALITY DURING ACUTE TOXICITY TESTS OF TECHNICAL CHLORDANE
Water
Dissolved oxygen
Total
Total
Specific
O-l
temperature,
Species °C mg/1
Fathead
minnow
Bluegill
Brook trout
Test 1
Test 2
Daphm'a magna
Test 1
24. 8a
+0.4
T23)
25.3
+0.2
T24)
14.7
+0.6
T24)
15.2
+0.5
ns)
20.9
+0.3
"(6)
6.5
+0.3
T33)
6.4
+0.3
n4)
7.5
+1.5
"(9)
6.6
+1.5
no)
6.8
+0.1
"(6)
„, alkalinity, Acidity,
saturation
77.1
+3.3
133)
76.5
+3.7
T14)
73.2
+14.9
"(9)
61.7
+16.8
"(10)
78.8
+2.3
"(6)
PH
7.70
+0.14
T28)
7.63
+0.10
(T2)
7.71
+0.41
"(9)
7.70
+0.14
no)
8.07
+0.04
no)
169
+1
(?8)
160
+2
(Tl)
155
+9
T9)
155
+4
(TO)
144
+2
(TO)
mg/1 CaC03
b
...
11.6
+2.8
"(9)
6.5
+1.8
HO)
6.7
+1.7
no)
hardness
152
+1
(?8)
161
+1
(IT)
149
+13
"(9)
135
+2
(TO)
143
+3
(TO)
' conductance,
umhos/cm
370C
+24
"(5)
393
+4
T2)
393
+26
"(3)
362
+4
T3)
372
+0
T3)
Continued ....
-------
TABLE 4. WATER QUALITY DURING ACUTE TOXICITY TESTS OF TECHNICAL CHLORDANE—continued
Water
Dissolved oxygen,
temperature, %
Species
Daphnia magn
Test 2
Hyallela
azteca
t
a
^«ta
20.8
+0.5
19)
15.5
+0.5
18)
mg/1
6.6
+0.6
16)
7.2
+0.3
(7)
saturation
72.9
+6.6
"(6)
71.4
+3.3
(7)
Total
alkalinity, Acidity,
PH
8.04
+0.09
16)
7.85
+0.18
H3)
149
+3
T6)
156
+4
03)
mg/1 CaCOg
5.4
+2.2
16)
6.0
+3.0
T13)
Total
hardness,
154
+8
T6)
144
+2
03)
Specific
conductance,
ymhos/cm
394
+9
T2)
363
+7
T4)
aMeans +1 standard deviation, and number of measurements made per parameter.
No observation.
€Spec1f1c conductance measurements were composites taken at each sampling time.
-------
TABLE 5. MEASURED CONCENTRATIONS OF TECHNICAL CHLORDANE IN
ACUTE TOXICITY TESTS
1/4
Test
species
Fathead
minnow
Bluegill •
Brook trout
Test 1
Test 2
Daphnia magna
Test 1
Test 2
Hyallela
azteca
No
measurements
per test Tank I
3 N.D.a
4 0.06
+0.3
5 N.D.
2 N.D.
3 N.D.
3 N.D.
4 N.D.
Tank II
12.3
+1.1
12.8
+3.1
12.4
+3.6
21.0
+0.6
10.4
+5.0
10.4
+4.2
35.3
+12.3
Chlordane
Tank II
20.0
+6.5
39.2
+7.2
17.8
+10.2
37.2
+3.5
16.5
+2.4
14.4
+5.2
66.7
+10.3
concentration,
I Tank IV
28.4
+14.5
59.1
+3.4
20.0
+7.2
52.9
+34.8
21.8
+2.6
20.8
+7.6
83.7
+11.9
yg/l
Tank V
34.1
+9.4
81.5
+0.1
34.2
+8.2
117
+63
28.4
+6.2
28.3
+11.5
115.2
+J3.5
Tank VI
53.4
+10.0
104.3
+9.8
41.0
+15.2
125
+104
33.9
+3.7
42.8
+16.1
161.3
+15.3
No technical chlordane detected.
-------
200
s
-------
CHRONIC TOXICITY TO FATHEAD MINNOWS
Water Quality and Chlordane Concentrations
Water quality during the chronic toxicity test is summarized in Appendix
Table 10. Since the concentrations of chlordane, acetone, and Triton X-100
had- no apparent influence on the water quality parameters measured, the data
for each week were pooled and mean values reported. Water temperatures were
approximately 21°C upon initiation of toxicant exposure, but were raised to
25°C during a 6-week interval and maintained within a degree of that tem-
perature for the duration of the experiment. Problems with the temperature
control apparatus prevented starting the test at 25°C. Dissolved oxygen
concentrations were maintained at greater than 60% of air saturation without
artificial aeration. Alkalinity, pH, and hardness levels of the ambient
water were very uniform.
Concentrations of technical chlordane, measured in the six tanks com-
prising each replicate, are summarized in Appendix Table 11. In general,
measured concentrations were 55% of desired. Although the reasons for the
loss were not ascertained, they could have been due to chlordane adsorption
to the surfaces of the test apparatus, to organic material, or because of
assimilation by the test organisms and epiphytes. Chlordane analysis of
hexane extracts of scrapings of algal-bacterial mats from the walls of the
tubes running between the diluter and the test chambers indicated that sub-
stantial amounts of the pesticide were associated with these organisms.
Measured aqueous concentrations of technical chlordane averaged 0.36 +_
0.16, 0.75 +0.25, 1.38 +0.57, 2.78 + 1.06, and 6.03 +2.25 yg/1. Traces
of chlordane were sometimes detected Tn control chambers.
Chronic Effects of Chlordane on Survival, Growth, and Reproduction
Under the aforementioned experimental conditions, fathead minnows
were cultured through one generation. Growth and survival of fathead min-
nows introduced as 1- to 5-day-old fry (termed f -generation) was very poor,
apparently because the food ("TetraMin") was not°small enough for all the
fry and possibly not of adequate nutritive value. Regardless of the quality
of the diet, chlordane may have slightly retarded growth of fry exposed for
the first 30 days to concentrations at and above 2.78 yg/1 (Table 6).
Analysis of variance of the growth data did not indicate that there were any
statistically significant differences between the controls and the treatment
groups at the 95% level of confidence. After 60 days, there were no apparent
differences in growth, leading to the conclusion that the insecticide had no
significant adverse effect at the concentrations employed.
At the end of the chronic test, minnows reared in 6.03 yg/1 chlordane
were significantly larger (p < 0.01) than controls (Table 7). However,
the importance of this difference is suspect because only eight individuals
remained in the high concentration at termination relative to 20 to 30 fish
in each of the other treatments.
39
-------
TABLE 6. TOTAL LENGTHS OF FATHEAD MINNOW FRY CHRONICALLY
EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone. ,
vg/1
Control
Control
0.36
0.36
0.75
0.75
1.38
1.38
2.78
2.78
6.03
6.03
30
F0-gen.a
9.7C
+2.6
8.4
+1.7
8.0
+1.2
8.8
+2.1
8.4
+1.4
8.5
+0.8
8.3
+2.1
8.3
+2.2
7.4
+1.2
7.6
+1.1
6.9
+0.9
7.4
+1.5
Total length
days
F^-gen.
13.3
+2.4
12.1
+1.9
11.4
+1.8
12.5
+2.4
11.1
*}•»
12.7-
+2.4
8.6
+1.5
10.3
+1.7
12.0
+2.1
11.8
+2.6
11.3
+2.6
11.4
+1.7
, run
60
Fo-gen.
12.3
+2.8
10.5
+2.8
12.9
+2.7
12.8
+4.3
12.2
+5.2
12.5
+3.7
12.0
+4.2
12.0
+3.6
10.7
+3.3
12.2
+4.7
9.5
+2.0
12.5
+3.4
days
F-j-gen.
21.4
+4.5
17.7
+3.2
19.1
+4.3
19.4
+4.4
20.3
+3.2
18.7
+3.9
18.2
+3.7
20.5
+4.2
19.1
+15.0
19.3
+4.6
19.8
+5.5
21.4
+4.4
aF -generation constitutes 1- to 5-day-old fry from parents having
no known previous history of chlordane exposure.
regeneration represents progeny spawned by adults having 6
months' chlordane exposure.
°Mean +1 standard deviation.
40
-------
TABLE 7. LENGTHSAND WEIGHTS OF ADULT FATHEAD
MINNOWS AT TERMINATION OF CHRONIC EXPOSURE
TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
ug/1
Control
Control
0.36
0.36
0.75
0.75
1.38
1.38
2.78
2.78
6.03
6.03
No. fish
14
15
15
15
13
13
15
17
8
13
3
5
Total length,
mm
46a
±5
49
±5
47
±5
53
±5
50
±8
53
±6
48
+6
48
+7
54
+8
50
+6
59
+6
56
+4
Wet body
weight,
g
1.2
+0.5
1.5
+0.7
1.0
+0.6
1.7
+0.6
1.3
+0.7
1.4
+0.7
1.1
+0.5
1.3
+0.7
1.7
+0.7
1.3
+0.7
1.9
+0.9
1.7
+0.4
aMean +_1 standard deviation. With the exception of one
of the controls, fish which died during spawning are
included in the summaries.
41
-------
Mortality of minnows was extensive during the first month, particularly
in the first 2 weeks, and was presumed to be due to an inadequate diet.
Although this was later corrected with a better diet, the poor early sur-
vival of the f -generation fry tended to obscure the relationship between
chlordane concentration and survival for the first 8 weeks of the test
(Table 8). Subsequent mortality, i.e. from 8 weeks up to the time of spawn-
ing at 24 weeks, was negligible, with fewer than three fish dying in any
treatment. Mortality during spawning was extensive, particularly in fish
exposed to chlordane concentrations greater than 0.75 yg/1. Although vari-
ation in mortality between treatments was considerable, it appears that
chlordane posed an additional stress on the fish at a time when they were
naturally stressed.
Details of spawning activity, embryo production, and hatching of fry
are given in Table 9. In general chlordane had no effect on the number
of spawnings, embryos produced per female, or the percentages of normal,
abnormal, or dead fry observed at hatching. Problems were regularly en-
countered with fungus on incubating embryos. In the majority of cases, _S.
parasitica on dead embryos spread to adjacent living ones despite daily
immersion for 5 min in a 4 mg/1 solution of zinc-free malachite green. This
problem occurred in all treatments including controls.
Subsequent growth of the f,-generation fry is tabulated in Tables 6
and 10. As also observed for f -generation fry, the second generation
progeny were slighly smaller after 30 days in 6.03 yg/1 chlordane than
the controls, although differences were not statistically significant.
After 60 days, there were no significant differences in size between fish
in the five chlordane concentrations and the control.
Mean mortality of the f.-generation through the first 60 days ranged
from 15 to 40% and was not increased by any of the chlordane concentrations
used (Table 10).
Statistical analysis of the results indicated that none of the concentra-
tions employed had any significant deleterious effects on any of the life
cycle stages of the fathead minnow. On the other hand, one apparent effect
from several of the chlordane concentrations was suggested, in that concen-
trations greater than 0.75 yg/1 caused increased mortality of adult minnows
during the period of spawning. Although this result is conceivable since
adults are already stressed and incur mortality naturally during spawning,
its significance can be challenged because of the high fry mortality that
occurred in all treatments during the first 2 months of the experiment.
Without repeating the test, the question of whether these fish were already
in a weakened condition will remain.
42
-------
TABLE 8. MORTALITY OF F -GENERATION FATHEAD MINNOWS
DURING CHRONIC°EXPOSURE TO CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.36
0.36
0.75
0.75
1.38
1.38
2.78
2.78
6.03
6.03
Cumulative percent mortality9
4
weeks
82
80
72
44
56
70
50
56
70
68
78
66
8
weeks
86
82
78
50
70
72
64
64
80
70
94
90
12
weeks
86
82
78
50
72
72
68
64
80
74
94
90
24
weeks
86
82
78
52
72
72
68
66
82
76
94
90
%
mortal ity
during
spawning0
6.6
0
13.3
26.7
23.1
0
40.0
93.3
75.0
15.4
33.3
20.0
aBased on a 50 fish per concentration.
Fish "thinned" at this time, which was just prior to the
commencement of spawning. Excess males were removed and
minnows of equivalent age from the culture facility were
used to bring the densities in the control tanks to 15
fish each.
cBased on 15 fish in each of the control and 0.36 yg/1 con-
centrations, and 26 fish (13 and 13) in 0.75 yg/1, 32 fish
(15 and 17) in 1.38 yg/1, 21 fish (8 and 13) in 2.78 yg/1,
and 8 fish (3 and 5) in 6.03 yg/1 chlordane at the begin-
ning of spawning.
43
-------
TABLE 9. SPAWNING HISTORY OF FATHEAD MINNOWS CHRONICALLY EXPOSED TO TECHNICAL
CHLORDANE
Measured concentration of chlordane, ug/1
Parameter Control Control 0.36 0.36 0.75 0.75 1.38 1.38 2.78a 2.78 6.03 6.03
No. females 97 11 688 12 7371 2
No. of
spawnings 30 16 25 12 16 10 23 14 2 19 6 10
Avg. No.
spawnings/ 3.33 2.29 2.27 2.00 2.00 1.25 1.92 2.00 0.67 2.71 6.00 5.00
female
No. eggs 4,960 1,262 3,788 627 1,949 487 2,970 1,550 20 1,926 527 576
Avg. No.
eggs/ 551 180 344 105 244 61 248 221 20 275 527 288
female
Avg. No.
eggs/ 165 79 152 52 122 49 129 111 10 101 88 58
spawning
Percent
hatch 37.5 27.3 60.1 29.1 72.5 38.2 55.7 40.0 2.8 46.0 44.5 54.9
Percent
abnormal fry 1.7 0.8 3.6 1.4 2.0 0.2 2.3 1.2 0 1.2 0.9 2.9
Percent
dead fry 3.7 1.8 4.6 6.1 12.2 0.1 3.7 4.9 0 6.7 3.0 5.2
aDue to complete mortality of mature males during spawning, males and females co-existed for only the
first 3 weeks of spawning.
-------
TABLE 10. MORTALITY AND RELATIVE SIZE
OF REGENERATION FATHEAD MINNOWS CHRONICALLY
'EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
ug/1
Control
0.36
0.75
1.38
2.78
6.03
After 60 days1
Mortality,
%
40a
+14
"(3)
23
+19
"(4)
15
+6
T3)
38
+16
"(3)
28
+17
T4)
28
+19
"(4)
exposure
Wet body
weight,
g
0.113b
+0.062
"(90)
0.094
+0.071
"(156)
0.096
+0.047
"(128)
0.106
+0.067
"(93)
0.095
+0.064
"(145)
0.117
+0.078
TI44)
*Mean +1 standard deviation and number of groups of 50
fry tested.
DWeighted mean +1 standard deviation and number of
individuals from which estimate made.
45
-------
CHRONIC TOXICITY TO BLUEGILL
Mater Quality and Chlordane Concentrations
The results of the water quality monitoring program for the bluegill
chronic are summarized in Appendix Table 12. Water temperatures were gradu-
ally adjusted from an initial level of 19°C to a temperature of 28°C, which
was required for induction of spawning. Thereafter, they were maintained at
28°C for the duration of the experiment. From the inception of testing, on
5 December 1972, until 31 March 1973, no aeration of the test chambers was
necessary. But as of the first of April 1973, the percentage saturation of
dissolved oxygen had dropped to below the required minimum of 60%, and
artificial aeration with oil-free compressed air was instituted. During the
experiment, the other variables remained relatively constant.
Concentrations of technical chlordane were measured in each set of
six treatments every other week. Since data collected prior to 26 March
1973 were considered unreliable because of analytical problems, they were
not included in the summary in Appendix Table 13. Mean measured concen-
trations ranged from 40 to 52% of desired and averaged 0.25 + 0.12, 0.54 +
0.21, 1.22 +0.53, 2.20 +0.56 and 5.17 +1.57 yg/1.
Chronic Effects on Survival, Growth and Reproduction
At the beginning of the chronic test (5 December 1972), 20 yearling
bluegill averaging 144 mm in total length and 58 g in wet body weight were
introduced into each of the 12 test chambers. During the 9.5 months of
continuous toxicant exposure, they grew an average of 19% in total length,
to 172 mm, and 78% in wet body weight, to 103 g (Table 11). Chlordane did
not have any statistically significant effect on growth of f -generation
bluegills at either 3, 5, or 9.5 months.
Aside from an anomalous mortality totalling 35% in one of the control
replicates, which was tentatively assigned to a brief outbreak of bacterial
hemorrhagic septicemia, bluegill mortality was largely confined to the 2.20
and 5.17 yg/1 concentrations (Table 12). Bluegill began dying in greater
numbers from 12 to 22 weeks in the 5.17 yg/1 concentration, but experienced
highest mortality during the period of spawning (22 - 37 weeks). Mortality
of fish exposed to 2.20 yg/1 chlordane was also confined largely to the 15
weeks of spawning activity. None of the fish exposed to toxicant concen-
trations lower than 2.20 yg/1 died. None of those which died between 12 and
22 weeks or during spawning showed evidence of erratic behavior or disease.
Moribund fish died passively. Bluegill held in the two highest concentra-
tions had greatly diminished appetites relative to the other groups.
Bluegill began spawning on 8 June 1973, 6 months after the beginning
of the chronic test. Embryo production was greatest in the control, 0.25
and 0.54 yg/1 concentrations, was substantially reduced in the 1.22 yg/1
level, and did not occur in the 2.20 and 5.17 yg/1 concentrations (Table
13). Hatching success ranged from 25.0 to 70.5% and did not appear to be
46
-------
TABLE 11. GROWTH OF F -GENERATION BLUEGILL DURING CHRONIC EXPOSURE TO
0 TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.25
0.25
0.54
0.54
1.22
Total length, mm
0
months
143a
+17
143
±16
142
+13
147
+19
143
+15
141
+14
146
+21
3
months
155
+22
158
±18
154
+18
158
+21
150
+.17
153
+15
160
+23
5
months
161
+19
163
+19
161
+15
163
+17
157
+14
159
+15
166
+20
9.5
months
167
+19
172
+24
173
+17
172
+19
174
+14
168
+20
169
+27
0
months
58
+27
58
+23
53
+15
63
+27
58
+22
53
+17
64
+28
Wet body weight, g
3
months
81
+33
81
+32
75
±24
85
+32
70
+24
75
±24
84
+34
5
months
99
+31
101
+36
94
+26
102
+33
87
+25
96
±33
107
+39
9.5
months
98
±38
104
±50
105
±29
103
+38
107
±30
102
±42
no
±58
Continued ..
-------
TABLE 11. GROWTH OF F -GENERATION BLUEGILL DURING CHRONIC EXPOSURE TO
0 TECHNICAL CHLORDANE—continued
oo
Meas.
chlordane
cone.,
vg/1
1.22
2.20
2.20
5.17
5.17
Total length, mm
0
months
146
±17
146
±18
145
±17
143
+11
147
±20
3
months
159
+17
158
±21
156
±21
153
+15
164
±24
5
months
160
+15
160
±16
162
±19
160
+18
166
±23
9.5
months
172
±16
179
±13
168
±22
178
+ 9
b
0
months
59
+21
58
±13
58
±25
57
+19
63
±30
Wet body weight, g
3
months
78
±26
79
±22
78
±34
69
+27
90
±34
5
months
92
±30
91
±28
100
±38
95
+35
100
+42
9.5
months
106
±37
98
±20
97
±52
106
±24
b
aMean ±1 standard deviation.
No fish remaining.
-------
TABLE 12. MORTALITY OF F -GENERATION BLUEGILL DURING
CHRONIC EXPOSURE TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.25
0.25
0.54
0.54
1.22
1.22
2.20
2.20
5.17
5.17
Cumulative percent mortality3
4
weeks
10.0
0.0
0.0
0.0
0.0
10.0
0.0
0.0
10.0
0.0
0.0
0.0
8
weeks
20.0
0.0
0.0
0.0
0.0
10.0
0.0
0.0
10.0
5.0
10.0
15.0
12
weeks
35.0
0.0
0.0
0.0
10.0
10.0
0.0
5.0
10.0
5.0
15.0
35.0
22
weeks
35.0
0.0
5.0
0.0
10.0
15.0
0.0
5.0
10.0
10.0
25.0
60.0
No. fish
remaining
after
thinning
8
10
10
10
10
9
9
10
10
10
8
7
01
h
mortality
during
spawning,
22-37 weeks
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
30.0
12.5
100.0
aBased on 20 fish per chamber. At thinning, numbers of fish were
reduced to a maximum of 10 per chamber.
49
-------
TABLE 13. SPAWNING HISTORY OF BLUEGILL CHRONICALLY EXPOSED TO TECHNICAL CHLORDANE
en
O
Meas.
chlordane
cone.,
yg/l
Control3
Control
0.25
0.27
0.54
0.54
1.22
1.22
2.20
2.20
5.17
5.17
No.
females
5
7
6
7
6
6
6
7
7
7
7
3
Mean No.
spawnings/
female
0.20
0.43
0.33
0.57
0.67
0.50
0.17
0
0
0
0
0
Mean No.
embryos/
female
20
2,252
400
3,559
3,442
2,074
263
0
0
0
0
0
Mean No.
embryos/
spawn
100
5,255
1,200
6,229
1,561
4,148
1,575
0
0
0
0
0
hatch
65.5
63.8
51.8
32.5
25.0
25.1
45.5
* K
66.5°
32. 5^
70.5°
% fry
Abnormal
1.0
1.7
0.6
1.1
0
0.6
0.7
0.5
1.0
7.5
1.5
Dead
0
0.8
0.9
0
0.2
0.3
0.7
0
0.5
7.5
0.5
Testes of male fish appeared to be largely immature.
Control eggs incubated.
-------
influenced by the levels of technical chlordane tested. Embryos spawned by
control bluegill and transferred to the 2.20 and 5.17 yg/1 concentrations
hatched in proportions which overlapped those of embryos spawned by fish in
the other treatments. However, there were slightly greater proportions of
dead and abnormal fry (i.e. erratic swimming behavior or structural defects)
at hatch in the 5.17 yg/1 concentration.
• When it had been determined that all bluegill had completed spawning,
they were killed, weighed, measured for total length, and the condition
and size of their gonads assessed and measured (Table 14). Although the
majority of both sexes had well developed and essentially unspent gonads,
those of males in one of the control replicates appeared to be immature
and weighed less than those of males from most of the other treatments.
Testes of bluegill exposed to 5.17 yg/1 were also generally smaller than
those of other male fish. Since these fish did not spawn, the effect of
successful spawning in reducing testicular mass was not a factor. In con-
trast to the differences in gonadal development noted for males, females had
well-developed ovaries. The ovaries of fish exposed to 2.20 yg/1 were some-
what smaller than other groups and these fish did not spawn. These observa-
tions suggest that the test conditions were not conducive to spawning or the
fish were not old enough. According to reports by others (D. T. Allison,
EPA, ERL-D, personal communication), most of the bluegill may have simply
been too young.
Few progeny were available for growth and survival studies, and the
results are considered inconclusive. Survival data, detailed in Table 15,
did not indicate treatment effects. Growth data suggested that control
fish grew less rapidly than those in the intermediate concentrations.
Only in the 2.20 yg/1 treatment was there a suggestion of diminished growth
(Table 16). All of the fry reared in 5.17 yg/1 chlordane died within 30
days.
In summary the most consequential effect of technical chlordane on blue-
gill was inhibition of reproduction at the 2.20 and 5.17 yg/1 levels.
Apparent but non-significant effects were noted on reproduction in the 1.22
yg/1 concentration and on hatching success in the lowest concentration tested,
0.25 yg/1. Bluegill did not spawn in one of the 1.22 yg/1 replicates and
hatching success of fry in chambers receiving chlordane was no greater than
80% that of controls. Thus, although adverse chronic effects are certainly
possible at lower concentrations, the 2.20 yg/1 was the lowest level at
which definite effects were observed.
CHRONIC TOXICITY TO BROOK TROUT
Water Quality and Chlordane Concentrations
During the 13-month duration of the chronic toxicity test utilizing
brook trout, there were only small fluctuations in water quality. Dissolved
oxygen concentrations averaged at least 60% of air saturation, although
artificial aeration was needed to maintain this level after the test had
been in progress for 2.5 months (Appendix Table 14).
51
-------
TABLE 14. CONDITIONS OF ADULT BLUE6ILL AT TERMINATION OF
CHRONIC TOXICITY TEST
Cn
Meas.
chlordane
cone.,
M9/1
Control
Control
0.25
0.25
0.54
0.54
1.22
Males
Total
length*
mm
188a
+8
202
±7
183
+.14
196
±3
187
+11
192
+6
195
+5
Wet
weight,
9
139
+25
169
+20
125
+33
157
+5
137
+27
155
+18
174
+9
Testes
weight,
mg
349
±178
2686
±297
439
±214
1331
±219
820
+640
933
±263
1924
±579
Testes
weight,
mg/g
2.4
±0.9
16.0
±4.5
3.4
±0.8
8.5
±1.6
6.2
±4.4
6.2
±2.3
15.4
±7.9
Total
length,
mm
154
±10
159
±15
166
±16
162
±13
165
±4
157
±9
156
±23
Females
Wet
weight,
g
73
±15
76
±22
86
±22
80
±12
88
±10
76
±16
78
±40
Ovary
weight,
mg
2538
±848
3609
±832
3970
±1605
3132
±1458
3920
±1700
3515
±1604
4282
±4296
Ovary
weight,
mg/g
34.1
±6.1
51.6
±19.5
46.1
±11.4
41.5
±23.5
44.4
±17.3
46.3
±17.6
47.9
±23.0
Continued ....
-------
TABLE 14. CONDITIONS OF ADULT BLUEGILL AT TERMINATION OF
CHRONIC TOXICITY TEST--continued
Meas.
chlordane
cone.,
yg/1-
1.22
2.20
2.20
5.17
5.17
Males
Total
length,
mm
188
+7
186
+8
200
+8
177
±9
b
• • •
Wet
weight,
g
152
±24
no
+12
173
+14
106
+24
• • •
Testes
weight,
mg
1607
+854
754
+748
747
+8
588
+446
• • »
Testes
weight,
mg/g
10.6
±5.9
6.9
+7.1
4.4
±0.4
5.0
±3.1
• • •
Total
length,
mm
165
±13
176
+14
155
±4
b
• • •
• • *
Females
Wet
weight,
g
86
+19
93
±22
67
+6
• • •
• • *
Ovary
weight,
mg
4016
±2259
3812
+2399
1373
±487
• • •
• • •
Ovary
weight,
mg/g
46.8
±25.4
39.3
±21.1
20.7
+7.3
• • •
• • •
Mean +1 standard deviation.
DA11 fish had perished.
-------
TABLE 15. SURVIVAL OF F,-GENERATION BLUEGILL
IN CHRONIC TOXICITY TEST OF TECHNICAL
CHLORDANE
Meas.
chlordane
cone.,
yg/l
Control
Control
Control
0.25
0.25
0.25
0.25
0.25
0.54.
0.54D
0.54
0.54
0.54
1.22
1.22r
2.20^
2.20^
2.20^
2.20^
5.17^
5.17C
Initial
No. fry
42
34
142
47
50
50
90
50
50
6
50
50
44
50
50
50
50
50
50
50
50
30 days
45.2
8.8
16.9
6.4
2.0
0
3.3
4.0
0
33.3
12.0
0
0
0
4.0
30.0
28.0
2.0
10.0
0
0
% survival
60 days
31.0
2.9
12.8
4.3
2.0
• • •
3.3
4.0
• • •
33.3
12.0
* • *
• • *
• • *
4.0
16.0
14.0
0
2.0
• * •
• • *
90 days
19.0
2.9
10.7
2.1
2.0
• • •
3.3
4.0
• • *
33.3
12.0
• • *
* * •
* • •
4.0
14.0
14.0
• • »
2.0
• • •
• » •
Fry transferred from 0.25 yg/1 concentration.
}Fry from eggs incubated in control.
'Fry hatched from control eggs.
54
-------
TABLE 16. GROWTH OF F,-GENERATION BLUEGILL DURING CHRONIC
TOXICITY TEST'OF TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.25
0.54
1.22
2.20b
30
No.
fry
23
24
8
8
2
35
days
Total
length,
mm
6.4a
+0.6
11.1
±1.8
13.0
+0.6
14.0
+2.6
11.5
6.7
+0.6
60
No.
fry
14
18
8
8
2
16
Growth
days
Total
length,
mm
9.9
+0.9
12.1
I1-7
28.0
+2.4
24.8
+2.4
27.5
14.9
+3.5
No.
fry
9
15
7
8
2
15
90 days
Total
length,
mm
17.3
+3.9
16.9
+2.0
36.8
+3.3
32.8
+4.0
36.0
18.7
+6.5
Wet
weight,
g
107
+35
64
+28
752
+206
481
+198
760
132
+88
Mean +1 standard deviation are given.
3Fry hatched from control embryos.
55
-------
As was observed in the chronic tests of the other fish species, mea-
sured chlordane levels were 42 - 58% of desired and ranged from 0.32 +_
0.18 to 5.80 + 2.15 yg/1 (Appendix Table 15).
Chronic Effects on Survival, Growth and Reproduction
Growth of f -generation brook trout throughout the chronic test did
not vary significantly between treatments in terms of either total length
or wet body weight, although fish exposed to 2.21 and 5.80 yg/1 chlordane
tended to be smaller at 3 months. At the beginning of the test, the trout
averaged 188 mm in total length and 70 g in wet weight. After 6.5 months,
they had increased 25% in length (to 248 mm) and more than doubled their
body weight (188 g). At termination average lengths and weights were 213 mm
and 281 g, respectively (Tables 17 and 18).
Prior to and during spawning, mortality was much higher among fish
exposed to 2.21 and 5.80 yg/1 than among controls or those exposed to the
lower (0.32 - 1.29 yg/1) concentrations (Table 19). For trout exposed to
5.8 yg/1 chlordane, mortality was 91.7% after 6.5 months' exposure and
complete at the conclusion of spawning. Control trout, which had been trans-
ferred to one of the 5.8 yg/1 tanks at thinning (the sole fish remaining
being transferred to the other tank), also died during spawning. Interest-
ingly, about half of the fish which perished had signs characteristic of
bacterial hemorrhagic septicemia (e.g. exophthalmia and lesions). Those
dying from what was believed to be chlordane poisoning were emaciated and
most exhibited impaired equilibrium for up to several weeks before death.
Convulsive or other behavior indicative of poisoning by some insecticides
was not observed.
After 8 months' exposure (28 November 1973), trout began spawning.
Total embryo production per female ranged from 62 to 400 in the control,
0.32, 0.66, and 1.29 yg/1 concentrations, but only from 0 to 47 in the 2.21
and 5.80 yg/1 concentrations (Table 20). Trout exposed to the two highest
chlordane concentrations also spawned fewer than 100 embryos at any time,
whereas spawns greater than 100 embryos each comprised from 9.1 to 26.9% of
the total spawns in the lower pesticide concentrations and the controls.
At the time spawnings were checked, i.e. within 24 hr of spawning,
the proportions of dead (opaque) embryos were greater at higher pesticide
concentrations (Table 20). On the average, only 8.0% of the embryos pro-
duced by controls were dead, compared to 14.8, 23.4, and 67.5% in the low,
mid-range, and high concentrations, respectively.
Spawns totalling more than 20 embryos were used for determination of
viability. Embryo viability was lower at higher technical chlordane con-
centrations, although embryos produced by one of the control tanks and one
of the 0.66 yg/1 tanks were nonviable. Average embryo viabilities declined
from 65% in the controls to 47% in the 0.32 yg/1 concentration to 17% in the
0.66 and 1.29 yg/1 concentrations (Table 21). None of the embryos produced
In the 2.21 and 5.80 yg/1 concentrations were viable. Four lots of 50
56
-------
TABLE 17. TOTAL LENGTHS OF F -GENERATION BROOK
TROUT CHRONICALLY EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.32
0.32
0.66
0.66
1.29
1.29
2.21
2.21
5.80
5.80
Total length, mm
0
month
188a
+8
185
+11
187
£13
192
±9
188
+15
187
+13
190
+15
195
±n
187
+10
186
+13
188
+9
187
+_15
3
months
211
±10
208
+14
208
+14
211
+10
210
+15
209
+14
210
+13
213
+12
195
+21
203
+17
209
+10
199
+27
6.5
months
250
f2
243
+16
249
+16
250
+10
246
+21
243
+22
252
+16
254
+15
257b
• • •
247
+21
241 b
• • •
21 Ob
• • •
12
months
!&
281
+22
278
±33
286
±19
286
+31
286
+11
•M»
285
+11
288
+14
b
• • *
279
+34
b
» • •
b
• • •
aMean +1 standard deviation,
bOne or no fish remaining.
57
-------
TABLE 18. BODY WEIGHTS OF F -GENERATION BROOK
TROUT CHRONICALLY EXPOSED TO°TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
vg/1
Control
Control
0.32
0.32
0.66
0.66
1.29
1.29
2.21
2.21
5.80
5.80
0
month
69a
+12
68
±12
68
+14
72
+12
72
+18
67
+15
74
+15
77
+13
71
+14
68
+16
69
+10
70
+17
Wet body
3
months
106
+18
103
+13
107
+20
105
f6
107
+21
104
+23
T07
+15
no
+16
90
±38
97
+25
96
+17
96
+31
weight, g
6.5
months
190
+33
177
+34
192
+42
190
+22
186
+_53
181
+50
196
+31
198
+36
21 Ob
• • *
179
+57
157b
• • *
107b
» • •
12
months
288
+65
268
+94
301
+78
276
+70
297
+149
278
+40
291
+38
286
+59
b
• • •
247
+100
b
• • *
b
• • •
+1 standard deviation.
One or no fish remaining.
58
-------
TABLE 19. MORTALITY OF F -GENERATION BROOK TROUT
CHRONICALLY EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
yg/1
Control
Control
0.32
0.32
0.66
0.66
1.29
1.29
2.21
2.21
5.80
5.80
Cumulative
3 6
mortality3, %
.5 mo
mo (up to thinning)
0
0
8
0
0
0
8
8
67
0
42
83
8
0
17
17
17
8
17
17
92
17
92
92
Mortal
ity
during spawning ,
6.5 - 1
Males
1/3
0/3
0/3
0/3
0/2
0/3
0/3
0/2
1/3
3/4
3/3c
0/0
2 mo
Females
1/4
0/4
0/4
0/4
0/5
0/4
0/4
0/5
1/2
1/2
4/4
2/2
Based on 12 fish per chamber.
Numerator and denominator of each expression equivalent to
number of fish dying out of total, i.e. 1/3 (male column)
indicates one of three males died during spawning.
Represent control fish transferred at thinning. The fish
which had survived to thinning in this treatment was trans-
ferred to the other replicate.
59
-------
TABLE 20. SPAWNING SUCCESS OF BROOK TROUT CHRONICALLY
EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
ug/1
Control
Control
0.32
0.32
0.66
0.66
1.29
1.29
2.21
2.21
5.80
5.80
No.
f emal es
4
4
4
4
5
4
4
5
2
2
4
2
Embryos/
female
290
90
400
62
215
153
259
126
47
30
32
0
No. embryos/ spawn
% embryos
>_! >20 >100 initially dead
22
9
29
15
16
10
14
29
10
5
16
• * •
11
3
14
4
12
6
5
8
1
1
1
4
1
4
0
5
2
4
1
0
0
0
7.3
10.1
13.9
21.1
25.3
5.1
6.6
50.9
40.9
13.3
67.5
60
-------
TABLE 21. VIABILITY AND HATCH OF EMBRYOS AND CONDITIONS OF F,-GENERATION BROOK
TROUT ALEVINS '
Meas.
cone.,
chlordane,
ug/1
Control
Control
0.32
0.32
0.66
0.66
1.29
1.29
2.21
2.21
5.805
5.80
Viability
No.
embryos
incubated
970
247
1,256
154
734
567
941
309
0
0
34 c
% viable
embryos
tank cone.
82.0 65 f
0 ™'*
42'7 46 7
79.8 4b>/
0 17 ,
39.3 "•'
23.1 ,7 4
0.3 "'*
0 0
Hatching success
No.
embryos % alev1ns % hatch
incubated normal abnormal dead tank cone.
450 97.3 2.4 0.3 91.8 gl>8
200 92.9 4.7 2.4 42.5 ,Q 7
100 100.0 0 0 94.0 oy
-------
embryos were transferred from control chambers to the 2.21 and 5.80 yg/1
concentrations, and viabilities for both treatments were as high (80 to 96%)
as controls, suggesting that the concentrations employed were not deleterious
within the 12-day period following spawning.
Hatching success was largely unaffected by technical chlordane up to
concentrations of at least 1.29 yg/1 (Table 21). Although too few eggs were
spawned by fish reared in the 2.21 and 5.80 yconcentrations to evaluate
hatching success, control eggs, incubated in these treatments for the 50 to 55
days (range of median hatch dates for all treatments) required for hatching,
survived as well (hatching success of 74 to 98%) as controls. Furthermore,
technical chlordane had no effect on the proportions of abnormally developed
or dead alevins at hatching (less than 3% in all cases).
Growth of the f,-generation generation progeny was followed over a
90-day period (Table 22). Upon hatching total lengths of subsamples of
alevins from each of the treatments were similar. After 30 days' growth,
total lengths of fry reared in 0.66, 1.29, 2.21 and 5.80 yg/1 tended to be
less than controls, but after 60 and 90 days, all chlordane-exposed fry were
larger than controls. Although analysis of variance indicated significant
differences after all three growth periods (p < 0.05), Dunnett's test indi-
cated that the significant differences were between the treatments and not
between the treatments and the control. Data on wet body weights suggested
similar relationships to those discussed for the total length data (Table
22).
Survival data for fry were incomplete, owing to high mortality in
approximately 20- to 40-day-old alevins which occurred on two successive
weekends. The cause was traced to chlorination of the water supply on
Fridays for removal of algae in storage reservoirs. The water supply to the
brook trout chronic test was not being passed through an activated charcoal
filter owing to the high volume flow required for this test. Lack of this
protection was responsible for the unanticipated mortalities. After the
cause of the mortality was identified, a solution of sodium t Mosul fate was
pumped into the water supply line at a concentration of 100 yg/1 to convert
chlorine to chloride ion. This eliminated further mortality.
In summary, chronic exposure of brook trout to technical chlordane
appeared to cause detrimental effects on survival, embryo production, spawn
size, and the viability and hatch of fj-generation progeny. However, the
importance of these effects can only be speculated since none were statis-
tically significant (p > 0.05). Survival, embryo production, and spawn size
were substantially lower than controls in insecticide concentrations greater
than 1.29 to 2.21 yg/1. Greater proportions of the embryos spawned were
found to be dead down to the lowest concentration tested (0.32 yg/1), while
12-day survival of the embryos was 29% lower in' this concentration than in
the controls. On the other hand, survival to hatching was reduced only
above 1.29 yg/1. From the above data it appears that the lowest concentra-
tion employed, 0.32 yg/1, would be deleterious to populations of brook
trout.
62
-------
TABLE 22. GROWTH OF F,-GENERATION BROOK TROUT DURING
CHRONIC EXPOSURE TO TECHNICAL CHLORDANE
Meas.
cone.
chlordane,
yg/1
Control
0.32
0.66
1.29
2.21
5.80
Total length,
At
hatch
14.4a
+0.6
T125)
14.5
+0.6
TH4)
13.8
+0.4
T25)
14.8
+0.4
T25)
14.5b
+0
T25)
14.6b
+0.6
T75)
30
days
21.4
+0.8
T89)
21.8
+0.8
T58)
17.2
+0.7
T24)
19.4
+0.6
T25)
18.6b
+0.9
T2D
17.4b
+0.7
T7D
60
days
28.4
+3.5
T39)
26.5
+1.8
T36)
29. 6 b
+2.1
T25)
29.4b
+2.0
T25)
• • •
• • •
iron
90
days
39.8
+2.9
T20)
44.7
+3.5
T24)
43.4b
+2.8
T14)
43.8b
+3.1
Tie)
• * •
• • •
Wet body
At
hatch
0.050
+0.005
"(110)
0.044
+0.004
160)
0.041
+0.003
"(19)
0;050
+0.002
"(12)
0.047b
+0.004
"(21)
0.050b
+0.006
"(57)
weight, g
90
days
0.610
+0.144
120)
0.910
+0.201
124)
0.803b
+0. 1 55
114)
0.847b
+0.169
"(16)
• • •
• • •
Means +_ 1 standard deviation and sample size are given.
'Control eggs that had been transferred to these concentrations.
63
-------
CHRONIC TOXICITY TO HYALLELA AZTECA
Mater Quality and Chlordane Concentrations
Water quality was measured eight times during the 9-week chronic test
utilizing H. azteca (Appendix Table 16). Water temperatures averaged 16.7
+ 1.0°C and" dissolved oxygen, 7.5 +_ 0.4 mg/1 (76% of air saturation).
A~s in all toxicity tests, the water was alkaline (pH of 7.86) and of inter-
mediate hardness (148 mg/1). Fluctuations from one week to the next were
small.
Measured chlordane concentrations were approximately 50% of desired,
averaging 1.41 +_ 0.77, 2.64 + 1.32, 5.32 +_ 3.24, 11.53 + 6.14, and 20.52
+_ 9.85 yg/1 (Appendix Table T7).
Toxicity
Two of the main indices of chlordane1 s chronic effects on H_. azteca,
namely growth and survival, were determined at the end of the experiment.
Use of only one point of measurement rather than several was selected be-
cause the responses of the animals to handling were unknown. Adult H_. azteca
are about one-quarter to one-third the size of Gammarus pseudolimnaeus, the
species for which this test was patterned; accordingly, it was presumed that
much greater care would be required during handling.
Survival of H. azteca was unaffected at technical chlordane concen-
trations less than 11.5 yg/1, where 92% or more of the specimens survived 9
weeks relative to 88 - 108% of controls (Table 23). However, survival was
significantly reduced to 12 - 36% in the 11.5 yg/1 concentration and to zero
in the 20.5 yg/1 level.
Growth of the amphipods was also affected by the presence of chlor-
dane. In both replicates, analysis of variance and Dunnett's test indi-
cated that amphipods exposed to 11.5 yg/1 chlordane were significantly
smaller (p < 0.05) than controls in terms of wet and dry weights (Table
23 and Appendix Table 18).
The chronic toxicity test indicated that growth and survival of H.
azteca were significantly reduced between concentrations of 5.3 and 1T.5
yg/1. The maximum acceptable toxicant concentration for technical chlor-
dane may exist within this range, but effects of this insecticide on
reproduction and on growth and survival of progeny should be examined before
arriving at this conclusion.
Accumulation of Chlordane
Contents of heptachlor, 3-, y- and "a"-chlordane, cis- and trans-
chlordane, and cis- and trans-nonachlor were determined on a dry weight
basis in H. azteca at the conclusion of the chronic test at 65 days. Con-
tents of each constituent increased with aqueous concentration. Concen-
tration factors tended to remain unaffected by the level of treatment for
64
-------
TABLE 23. RELATIVE SURVIVAL AND GROWTH OF HYALLELA AZTECA EXPOSED TO TECHNICAL
ON
Ol
CHLORDANE
Measured concentration of
Parameter
Replicate 1
No. survivors3
% survivors
Wet body weight, mg
Dry weight, mg
Replicate II
No. survivors
% survivors
Wet body weight, mg
Dry weight, mg
Control
27
108
6.3
+1.3
1.58
22
88
7.5
+1.3
1.92
1.4
23
92
6.2
+1.5
1.49
25
100
5.8
+1.3
1.55
2.6
23
92
6.4
±1-2
1.57
25
100
5.8
+1.6
1.53
technical chlordane, yg/1
5.3
24
96
5.1
+0.9
1.37
24
96
5.5
+1.6
1.35
11.5 20.5
3 0
12 0
3.8 ...
+0.7
0.87
9 0
36 0
D • o • • •
+1.0
f • oO • • •
25 individuals introduced initially per chamber.
3Average calculated weight per individual.
-------
heptachlor, the chlordenes, and cis-nonachlor, and increased only slightly
for c_[s_-chi ordane, trans-chlordane and trans-nonachlor. The highest con-
tents of each constituent were 357 yg/g for heptachlor, 92.3 yg/g for the
chlordenes, 260 yg/g for trans-chlordane, 220 yg/g for c_is_-chl ordane, 71.8
yg/g for trans-nonachlor, and 46.4 yg/g for cis-nonachlor (Table 24).
Net concentration of all constituents was very extensive. Even though
heptachlor was concentrated to a lesser extent than the other compounds, its
concentration factors were still quite high (16,700 to 31,040). Although
the cis- and trans-nonachlors comprised only 2.8 and 5.1% of the technical
chloHane, their storage was proportionally greater than all other major
constituents including the cis- and trans-chlordanes, which comprised 43% of
the insecticide. The compound accumulated to the greatest extent was cis-
nonachlor, for which concentration factors ranging from 95,030 to 144,TOlT
were calculated. The propensity of the components to be concentrated in-
creased in the following order: heptachlor, the chlordenes, trans-chlordane,
cis-chiordane, trans-nonachlor. and cijs_-nonachlor (Table 24).
The proportions of the chiordane constituents present in the amphipods
were different from those characterizing the neat insecticide, suggesting
differences in water solubility, uptake, or metabolism. For example, the
ratio of cis- trans-nonachlor in the neat technical insecticide was 0.55:1,
but the average ratio in the organisms was 0.71:1, indicating an enhanced
accumulation of cis-nonachlor relative to the trans isomer. A preferential
storage of cis-chiordane relative to that of the trans isomer was also
apparent, wTEF the mean ratio of cis- trans-chlordane of 0.85:1 in the tis-
sues being somewhat greater than tTiat (0.79:1) characterizing the neat
insecticide. There was also a diminution in heptachlor storage relative to
the two chlordanes, for the ratio in the tissues (0.07:1) was only 32% of
that in the stock formulation (0.23:1). Considerably more cis- and trans-
nonachlor were stored relative to cis- and trans-chl ordane."^Tie ratio of
chlordanes to nonachlors was 4.1:1 in amphipod tissues and 5.4:1 in the neat
insecticide. Although these studies were not intended to elucidate the
in vivo fate of the various chlordane components, it appears that the nona-
cFlors may have been particularly susceptible to uptake and deposition or
were generated in part through metabolism of such constituents as cis-
and trans-chlordane or heptachlor, which were present in diminishecTpropor-
tions in amphipod tissues.
The relative proportions of some of the constituents also appeared
to change as a function of aqueous technical chlordane concentration.
For example, the heptachlor/cjsj- and trans-chlordane ratio declined from
0.085:1 in specimens exposed to 1.4 yg/1 technical chlordane to 0.063:1
in those exposed to 11.5 yg/1, while the cis-/trans-nonachlor ratio declined
from 0.83:1 in amphipods exposed to 1.4 ygTT to 0.59:1 in those exposed to
11.5 yg/1. In contrast, the contents of the chlordanes relative to the
nonachlors may have increased since the ratio between them was slightly
Greater (4.2:1) in the two highest concentrations than in the two lowest
3.76:1 and 3.79:1). The cis-/trans-chlordane ratio was essentially con-
stant between treatments (0.83:1 to 0.87:1). These differences suggest that
amphipods which were exposed to the higher technical chlordane concentrations
66
-------
TABLE 24. CONTENTS AND CONCENTRATION FACTORS (C.F.) OF CHLORDANE CONSTITUENTS IN DRIED HYALLELA
ON
AZTECA THAT
HAD BEEN EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone.,
ug/1
Control
Control
1.4
1.4
2.6
2.6
5.3
5.3
11.5
11.5
Heptachlor
Content ,
, ug/gb
0.3
0.5
3.4
3.1
5.3
5.8
10.9
11.7
19.2
35.7
C.F,
c
* * «
24,290
22,140
20,390
22,310
20,570
22,080
16,700
31,040
Chlordenesa
Content,
ug/g
1.4
1.4
10.1
8.2
17.8
17.7
29.6
32.6
60.7
92.3
C.F.
* • •
55,500
45,060
52,660
52,370
42,960
47,320
40,600
61,740
C1s-chlordane
Content,
ug/g
1.9
2.7
19.2
16.2
36.9
35.5
73.5
81.7
176.9
220.0
C.F.
...
72,180
60,900
74,700
71,860
72,990
81,130
80,960
100,690
Trans-chlordane
Content,
ug/g
2.2
2.9
22.1
18.7
42.5
41.3
88.6
97.4
216.0
259.9
C.F.
• • •
65,770
55,660
68,110
66.190
69,650
76.572
78,261
94,170
C1s-noi
Content,
ug/g
0.2
0.6
5.3
3.8
9.2
9.1
14.9
16.8
30.6
46.4
lachlor
C.F.
• • •
135,200
96,940
126,370
125.000
100,400
113,210
95,030
144,100
Trans -ni
Content,
ug/g
0.5
0.5
6.1
4.9
11.8
11.4
23.2
26.1
58.9
71.8
onachlor
• • •
85,430
68,630
88,990
85,970
85,830
96,560
100,430
122,420
'Consisting of Y - B Peaks (13) and "a" peak (12).
ug/g residue per dry body weight.
Concentration factors (C.F.) not calculated for controls since on only one occasion was technical chlordane
detected.
-------
may have dealt with the various constituents differently than those exposed
to lesser concentrations.
CHRONIC TOXICITY TO DAPHNIA HAGNA
Water Quality and Measured Chlordane Concentrations
The standard battery of water quality parameters was measured four
times, just prior to and during the course of the chronic toxicity test
using D. magna. The water temperature averaged 20.9 +_ 0.5°C and the water
quality was very similar to that described earlier for the other chronic
toxicity tests (Appendix Table 19).
Desired concentrations of technical chlordane ranged from 6.1 to 96.9
yg/1, excluding the control, and were set high because of excessive loss of
the insecticide under the conditions of limited toxicant renewal. The
concentrations existing during the test ranged from 1.7 +_ 0.1 to 21.6 +_
9.6 yg/1 for the five treatments (Appendix Table 20).
Effects on Survival, Growth and Reproduction
Survival of the cladocerans from first instars to adults was poor
regardless of treatment. After 1 week, control survival averaged 80%, but
declined to 30% in the fourth week (Table 25). Survival of daphnids in
chlordane was essentially the same as that of the controls, except for those
exposed to 21.6 yg/1 chlordane, where only one of the initial 20 specimens
survived to the middle of the fourth week.
Growth of the cladocerans was determined only for instars produced
in the fourth week. Since dry weights of instars produced in the various
chlordane solutions were commensurate with those for controls, there did not
appear to be any adverse effects upon growth (Table 26).
Reproduction of Daphnia was highly variable and was apparently unaf-
fected by any of the concentrations of technical chlordane used (Table 25).
On the basis of the above data, which should be considered preliminary
pending completion of experiments having good control survival and repro-
duction, it appears that the only toxicologically effective concentration
was 21.6 yg/1, which had killed all first generation daphnids by the end of
the fourth week.
Accumulation of Chlordane
Accumulation of the major components of chlordane in D. magna was
similar in magnitude to that of H. ajteca, even though the cladocerans
were exposed for a maximum of 1 week and the amphipods for 2 months.
Uptake and storage are evidently very rapid in D. magna.
First instars produced in the last 7 days of the chronic test were used for
for residue analysis.
68
-------
TABLE 25. SURVIVAL AND REPRODUCTION OF DAPHNIA MftGNA
IN CHRONIC TOXICITY TEST OF TECHNICAL CHLOR~DATiF"~
Week
5/8/74
5/15/74
5/23/74
Meas.
chlordane
cone.,
yg/1
Control
Control
1.7
1.7
2.5
2.5
6.2
6.2
12.1
12.1
21.6
21.6
Control
Control
1.7
1.7
2.5
2.5
6.2
6.2
12.1
12.1
21.6
21.6
Control
Control
1.7K
1.7b
2.5
2.5
6.2
6.2
12.1
12.1
21.6
21.6
Adult
survival ,a
%
80
80
90
80
70
80
60
70
90
90
50
100
50
40
80
70
40
70
20
50
80
80
0
90
40
30
70
40
40
70
20
50
70
80
0
40
Production
Total
instars
0
0
0
0
0
0
0
0
0
0
0
0
25
16
13
7
13
34
7
13
39
27
0
36
12
22
176
3
28
334
28
91
74
220
0
15
Instars/avg.
No. adults
0
0
0
0
0
0
0
0
0
0
0
0
5
4
2
1
3
5
4
3
5
3
0
4
3
7
25
1
7
48
14
18
11
28
0
3
69
-------
TABLE 25. SURVIVAL AND REPRODUCTION OF DAPHNIA MAGNA
IN CHRONIC TOXICITY TEST OF TECHNICAL CHLORDAlJE^continued
Week
5/30/74
Meas.
chlordane
cone. *
yg/1
Control
Control
1.7
1.7
2.5
2.5
6.2
6.2
12.1
12.1
21.6
21.6
Adult
survival,3
Total
Production
Instars/avg.
% instars No. adults
40
20
60
0
40
70
20
50
60
70
0
0
115
57
804
» * *
215
619
162
166
143
791
0
85
29
29
134
...
54
88
81
33
24
105
0
170
aTen Daphnia initially introduced into each test container.
Values represent percentage of specimens remaining at the
end of a given week.
Accidental discard of remaining adults in this concentration.
70
-------
TABLE 26. AVERAGE DRY BODY WEIGHTS OF FIRST
INSTAR DAPHNIA MAGNA PRODUCED DURING FOURTH
WEEK
Meas.
chlordane
cone.,
vg/1
Control
Control
1.7
1.7
2.5
2.5
6.2
6.2
12.1
12.1
21.6
21.6
OF CHRONIC TOXICITY
CHLORDANE
No.
specimens
115
57
804
0
215
619
162
166
143
791
0
85
TEST OF TECHNICAL
Average dry
weight/individual,
pg
49.1
27.8
31.2
* • •
36.7
29.5
28.3
23.4
24.4
27.5
• * •
43.5
71
-------
As shown in Table 27, which gives the actual tissue levels of the
various components as well as their individual concentration factors, hepta-
chlor residues were lowest (range of 0.9 - 27.9 yg/g) and trans-chlordane
residues highest (9.6 - 370 yg/g) of the six components selected for analy-
sis. Daphnids tended to preferentially concentrate more cis-and trans-
nonachlor and less heptachlor and chlordenes than cis- or trans-chlordane.
Tissue residues of each compound appeared to be directly proportional to the
aqueous concentration of technical chlordane to which the animals were
exposed. The linearity of uptake and storage was corroborated by the uni-
form factors for each component, which varied little as a function of treat-
ment, except for the low aqueous concentration, where concentration factors
were considerably lower. Concentration factors varied from 5,290 to 12,900
for heptachlor to 32,000-144,850 for trans-nonachlor. The order of increas-
ing bioconcentration was: heptachlor, the chlordenes, trans-chlordane,
cis-chlordane, cis-nonachlor, and trans-nonachl or.
As was observed for amphipods, the relative proportions of the chlordane
constituents differed from those characterizing the neat insecticide. There
was preferential deposition of the cis- and trans-nonachlors and a rela-
tive diminution of heptachlor and the cis- and trans-chlordanes.
CHRONIC TOXICITY TO CHIRONOMUS NO. 51
Two partial chronic toxicity tests were conducted with Chironomus No.
51 to delimit "safe" and "unsafe" concentrations. The first test was pre-
liminary, limited to introduction of 25 newly-hatched larvae into each
of five insecticide concentrations and a control. The second test utilized
50 newly-hatched larvae and 12 test chambers, comprising five concentrations
and a control in duplicate.
Water Quality and Chlordane Concentrations
Levels of each of the water quality parameters comprising the standard
battery were essentially the same in both experiments. Concentrations
of dissolved oxygen were 80% of air saturation, pH levels averaged 7.94
and 7.90, and total hardness averaged 154 and 150 mg/1 CaCOv respectively
(Appendix Table 21).
Measured concentrations of technical chlordane ranged from 1.0 + 0.1
to 24.9 + 16.8 yg/1 in the first test and from 0.7 + 0.1 to 15.5 +_ 377 yg/1 in
the second (Appendix Table 22).
Toxicity
Adult Chironomus No. 51 emerged in the first test 11 to 15 days after
their introduction as newly-hatched larvae. All control larvae and those
held in 1.0 yg/1 emerged, but none of those reared in 2.4 to 24.9 yg/1 did.
The time to 50% adult emergence was 12.5 to 13 days for both treatments
(Table 28). Males, which constituted only 28-32% of all adults, tended to
emerge 1 day earlier (50% emergence in 11 days) than females.
-------
TABLE 27. CONTENTS AND CONCENTRATION FACTORS (C.F.) OF CHLORDANE CONSTITUENTS IN DRIED
DAPHNIA MAGNA THAT HAD BEEN EXPOSED TO TECHNICAL CHLORDANE
Meas.
chlordane
cone. ,
pg/i
Control
Control
1.7,
1.7d
2.5
2.5
6.2
6.2
12.1
12.1
21.6,
21. 6d
Heptachlor
Content,
ug/gb
0.5
0.5
0.9
2.3
1.9
3.0
9.8
7.4
9.8
27.9
C.F.
c
5,290
9,200
7,600
4,840
15,810
6,120
8,100
12,920
Chlordenes3
Content,
ug/g
5.7
4.3
4.8
8.5
9.2
11.8
36.9
26.1
31.6
72.5
C.F.
• • •
21,720
26,150
28,310
14,640
45,780
16,590
20,089
25,820
C1s-chl(
Content,
yg/g
6.8
6.1
8.9
27.7
26.6
71.8
126.0
124.3
152.0
333.8
jrdane
C.F.
* • •
27,550
58,320
56,000
60,950
106,960
54,070
66,120
81,340
• * •
Trans-ch'
Content,
ug/g
6.7
6.0
9.6
29.5
29.8
77.0
135.6
137.5
168.0
369.8
lordane
C.F.
• • •
• • •
23,530
49,170
49,670
51,750
91,129
47,350
57,850
71,340
Cis-noi
Content,
vg/g
1.5
1.4
2.2
5.4
5.9
16.7
30.4
28.4
29.6
52.2
lachlor
C.F.
...
46,220
77,140
84,290
96,200
175,120
83,830
87,370
86,310
Trans-ni
Content,
pg/g
1.6
1.5
2.8
9.7
7.7
27.8
45.8
44.2
62.7
125.5
Dnachlor
C.F.
• • •
32,300
76,080
60,390
87,920
144,850
71,630
101,600
113,930
Consisting of Y - 6 peaks (13) and "a" peak (12).
yg/g dry body weight.
Concentration factors not calculated for controls since technical chlordane was undetected.
No instars produced in fourth week.
-------
TABLE 28. CHRONIC EFFECTS OF TECHNICAL CHLOROANE ON CHIRONOHUS NO. 51
Parameter
Total adult
emergence
% emergence
% males
% females
Median emergence
time, days
Control
25
100
33
67
13
Test 1
1.0 ug/1
25
100
28
72
12.5
Test 2
2.4 ug/1 Control
0 11
0 22
63
37
13
Control
24
48
63
37
16
0.7 pg/1
10
20
44
56
17
0.7 pg/1 1.7 ug/1
11 0
22 0
36
64
15
1.7 pg/1
0
0
...
...
...
-------
In the second experiment, adult midges emerged only from the control
and low concentration (0.7 yg/1) and were not observed in the 1.7 to 15.5
yg/1 treatments (Table 28). Although adults were observed on the same day
(day 11) as in the first test, emergence was inexplicably spread over a
longer period (11 to 25 days) and median times to emergence were longer. In
the two control chambers, 50% of the adults had emerged 12 and 16 days,
respectively after their introduction as larvae. Fifty percent of the
midges exposed to 0.7 yg/1 chlordane emerged after 17 and 15 days.
Survival to emergence was much poorer in the second experiment than
in the first. Of the 50 larvae originally introduced into each chamber,
only 22-48% of the controls and 22-24% of those exposed to 0.7 yg/1 chlor-
dane survived. Larvae were observed 4 days after introduction in the
1.7 yg/1 concentration, but not thereafter. There was no evidence that
chironomids survived for even a short time in the 3.3 to 15.5 yg/1 con-
centrations.
In contrast to the first experiment where females predominated, more
males (60 to 63%) emerged in the controls than in the 0.7 yg/1 concentration
(36 to 44%) in the second test. Males tended to emerge earlier in the con-
trol chambers than females, but in the 0.7 yg/1 concentration, they emerged
at the same rate. Of the first 50% of the control midges to emerge, 80 to
92% were males.
On the basis of the results of the two experiments, technical chlor-
dane concentrations above 0.7 to 1.0 yg/1, i.e. 1.7 yg/1, were clearly
unsafe because they were lethal to the developing larvae. Complete life
cycle tests encompassing reproductive and hatching success, etc, would be
needed to ascertain whether lower levels are deleterious.
Accumulation of Chlordane
No residues of technical chlordane were detected in extracts of dried
adult Chironomus No. 51 and there was no evidence of the presence of
oxychlordane.
75
-------
SECTION VII
DISCUSSION
ACUTE TOXICITY TESTS
Chlordane appears to be generally less toxic in the short-term to
freshwater fish and invertebrates than endrin, dieldrin, aldrin, and DDT,
but more toxic than methoxychlor, lindane, benzene hexachloride (BHC) and
Guthion . In the present study continuously-renewed solutions of technical
chlordane were lethal in 96 hr to three fish species between 37 and 59 yg/1.
These values agree well with the data of Katz (23), Henderson et al. (22),
and of Macek et al. (25), but are consistently higher than values reported
by Konar (26) and lower than those given by Ludemann and Neumann (30). For
example, Katz (23) reported 96-hr LC50 estimates of 44 to 57 yg/1 for three
species of salmonids. Our estimate of 47 ug/1 for brook trout was within
this range. The 96-hr LC50 of 77 yg/1 at 23.8°C for bluegill reported by
Macek et al (25) was somewhat higher than we found (59 yg/1) for the same
species, but both of these estimates were above the 16.5 yg/1 LC50 estimate
given by Henderson et al (22). In general brief exposure to chlordane
would appear to be lethal to many fish species within the concentration
range of 1 to 100 yg/1. More than half of the toxic responses of fish
to chlordane reported in the literature (Table 1) and determined in this
project were within this range.
Acute toxicity tests conducted with continuous toxicant renewal would
in many cases be expected to result in lower lethal limits than tests con-
ducted without renewal (e.g. static conditions) because toxicant concen-
trations would not decline due to assimilation by the test organisms or by
sorption to debris or to the walls of the test vessel. Use of measured
rather than expected insecticide concentrations in estimating median re-
sponse limits should also improve their validity. While these arguments are
valid, the LC50 values obtained by us were not demonstrably lower than those
reported by others, as indicated above. Differences between static and
flow-through test results for chlordane may come to light when exposures are
extended beyond approximately 96 hr. For example, the data of Henderson et
al. (22) and of Katz (23) indicate that median lethal thresholds, the con-
centration at which lethality to 50% of the specimens ceases, were approached
or reached within 96 hr for fathead minnow, goldfish (Carassius auratus),
rainbow trout, and Chinook salmon (Oncorhynchus tshawytscha). In our stu-
dies a median lethal threshold was attained only with fathead minnows, but
only after 168 hr; toxicity curves for brook trout and bluegill were linear.
The absence of median lethal thresholds for exposures less than 96 hr would
76
-------
be expected if the poison had a cumulative action—which chlordane apparently
does—and if toxicant concentrations remained relatively constant for the
duration of the test.
Comparison of the toxicity test results for D_. magna and IHL azteca
with those reported in the literature for similar species indicates rela-
tively good agreement. The two 96-hr LC50 values for D. magna of 28 and
35 yg/1 were only slightly higher than the 48-hr LC50 of 20 yg/1 for the .
cladoceran Simocephalus serrulatus (6). Hyallela azteca are decidedly
less sensitive to chlordane than Gammarusjacustris. The 96-hr LC50 of
26 yg/1 for the latter species (35) was only 25% that for H. azteca, which
were exposed for a longer (168 hr) period. Although there is a great range
in the levels of chlordane reported to be toxic to aquatic invertebrates,
their sensitivity to this insecticide appears to be of the same order as
that for fish (i.e. acute lethal range of 1 to 100 yg/1).
CHRONIC TOXICITY TESTS
The lowest aqueous concentration of technical chlordane which we found
to have marked deleterious chronic effects was 0.32 yg/1, which lowered
brook trout embryo viability. This apparent "unsafe" level for chronic
exposure was less than 1% of the 96-hr LC50 for this species. Prominent
chronic effects were observed for bluegill and the chironomid at concentra-
tions around 2 yg/1. High mortality prior to and during the spawning period
and failure to spawn were the salient responses of bluegill that had been
exposed to 2.2 and 5.2 yg/1 chlordane. Larval mortality, which accounted
for the failure of adult emergence, was the main effect of 1.7 yg/1 chlordane
on Chironomus No. 51. Of lesser apparent sensitivity were fathead minnows,
daphnids, and the amphipod. These species were unaffected by chlordane
concentrations lower than about 5 to 10 yg/1.
Although the chronic toxicity tests we conducted produced much needed
information on the effect of this insecticide on growth, survival, and
reproduction of several fish and invertebrate species, they failed in every
case to produce hard, unequivocal data on what concentrations were detri-
mental and which were not for all major life stages of each species. Con-
sequently, technical chlordane may ultimately be found to be more toxic to
these species than reported here. For example, the brook trout and bluegill
experiments were partial rather than full life cycle tests because they
were begun with yearlings instead of fry or embryos. It is quite conceiv-
able, accepting that chlordane's toxicity is cumulative, that greater effects
on the f -generation would have been observed had younger specimens been
used. Further, neither the trout nor bluegill tests fully evaluated effects
on f,-generation progeny. In-both experiments, poor survival compromised
interpretation of results. The most notable liability of the fathead minnow
chronic was the poor early survival of the fry. As stated earlier, the
question of whether the surviving fish were in a weakened condition will
remain, even though there is the possibility that the survivors represented
the most fit individuals because the weaker fish were selected out. Simil-
arly, poor survival—and hence reproduction—of f -generation daphnids made
77
-------
conclusions tenuous on the extent of effects. Finally, while the chironomid
and amphipod tests went well, they did not evaluate toxicant effects on the
f-j -generation.
.Chronic toxicity, life cycle tests represent an important advance
in the sophistication and sensitivity of aquatic toxicity testing. Though
costly and time-consuming, they are probably the best means for directly
estimating cumulative, long-term effects on most developmental stages of an
organism. Because emphasis is placed, when possible, on the results of such
tests in setting water quality standards, it is important that the tests be
standardized to some extent to insure maximum utility and validity of results.
This standardization could include Improvements in test conditions to achieve
better and more uniform control of specimen quality, identification of key
response; parameters, and recommendations as to acceptable statistical analy-
ses. For the full potential of the statistics to be realized, several of
the chronic tests require better design. Most notable is the need for addi-
tional replicates for assessing the various responses of the f -generation.
Increasing the replicates from two to perhaps four, for example, would increase
the within-treatment degrees of freedom from 1 to 3 (for a simple one-way
analysis of variance). With the present design, differences often have to
be rather astounding to be significant because there is considerable within-
treatment variation. Also, it would be desirable to stipulate minimum
replication for the viability-hatching success determinations since there is
the possibility that more data will be accumulated than necessary. Since
few laboratories have resident biometricians, information could be wasted if
the experimental design were left solely to the discretion of the investi-
gator. This would be particularly true for the first few tests conducted.
In addition to the fundamental changes suggested above, there are
specific changes in methodology which should be considered for each of
the organisms we tested. The apparent lack of fertilization of spawned
eggs in the trout tests was anomalous and should be studied further. It
was also encountered in three other brook trout tests we conducted concur-
rently for a separate project (63). Possible reasons for the infertility,
which appeared to be random, include physical inability to spawn because the
substrates were too small or behavioral changes caused by fish density or
the nature of the heirarchal relationships. It is also unknown whether all
trout from this stock reach reproductive maturity in 2 years.
In the bluegill test, the fish were probably too young to spawn exten-
sively. Better results might be achieved by beginning the test with 2-yr-
old specimens. Improved techniques for incubating embryos and rearing fry
should be developed which Include verified methods of disease control and
proven diets. Until the fry can consume brine shrimp nauplii, it would be
advisable to feed them laboratory cultures of rotifers, for example, instead
of "green" water because the latter might contain parasites and pathogens.
Also, maintenance of high food densities, such as cultures of rotifers in
phytoplankton, might cause a significant proportion of the toxicant to be
sorbed to or assimilated by the food. This could alter the mode of intoxi-
cation if not the toxicant concentration. Finally, some consideration
78
-------
should should be directed to the adequacy of the bluegill spawning substrates
and the validity of spawn size estimates, for embryos are often spread about
the adult tank, bound to debris, and eaten prior to being checked by the
investigator. While the substrates were acceptable to the adults, it was
difficult to remove the embryos with a fine brush. To our knowledge, no one
has carefully evaluated different methods of embryo removal with reference
to the injury they cause.
The conduct of chronic tests with fathead minnow is fairly routine with
proper experience, and the only improvement which is recommended is to
gain a better fix on the type and concentration of chemicals used for con-
trolling fungus during embryo incubation.
In the test using cladocerans, it might be better to separately assess
growth because there are not enough f -generation specimens available in the
recommended procedure (60) and f,-generation instars produced with a given
week will of course differ in age.
In retrospect, Myall el a azteca and Chirpnomus No. 51 are not the most
desirable species for chronic tests. Newly-hatched H^. azteca are really too
small for rapid enumeration or capture and would hamper a hatching success
determination. A species such as Gammarus lacustris would be more desirable
because it is larger (around 20 mm in adults) and quite widespread through-
out the United States. Chironomus No. 51, in addition to the liability that
it has not been taxonomically described, has the embryos helically arranged
within the skein, which makes it very time-consuming to accurately count and
separate embryos for a hatching success determination.
ACCUMULATION OF CHLORDANE
Technical chlordane was accumulated extensively in H_. azteca and D.
magna, but not at all in the winged adults of Chironomus No. 51. For Foth
amphipods and daphnids, tissue concentrations of a particular component were
fairly proportional to aqueous concentration. However, both species tended
to concentrate the various compounds to different extents. Concentration of
the components was comparable between amphipods and daphnids, except that
amphipods concentrated at least 2-times more of the chlordenes and hepta-
chlor than daphnids.
The absence of notable residues of technical chlordane in adult chiro-
nomids exposed to both 0.7 and 1.0 yg/1 chlordane in separate experiments is
difficult to interpret. Recent studies of DDE uptake by fourth instar £.
tentans by Derr and Zabik (68) indicate a passive mode of uptake and an
essentially linear relationship between aqueous and tissue DDE concentration.
Similar findings have been reported earlier by Kerr and Vass (69). Although
Chironomus No. 51 may possess efficient mechanisms for metabolizing or
excreting technical chlordane components at all developmental stages or
during the transition from larva to adult, additional experiments designed
to monitor the water-tissue concentration relationships for all develop-
mental stages and several toxicant concentrations are needed to clarify the
79
-------
somewhat anomalous results we obtained for this species.
For both amphipods and daphnids, comparisons of the proportions of
the chlordane components in the tissues with those in the neat material
indicated that most were stored in different proportions. Cis- and trans-
nonachlor were stored to a greater extent in both species than the other
components. Similar findings have been made for fish collected from Lakes
Superior and Huron (personal communication, L. Mueller, EPA, ERL-D). Since
there appeared to be little if any compositional change relative to the neat
material when technical chlordane was measured in aqueous solution, these
components were either taken up preferentially, were metabolites of other
constituents, or were particularly refractory to metabolism. The lower
tissue ratio of heptachlor to cis- and trans-chlordane and that of the
chlordane isomers to the nonachlor isomers suggests that these compounds may
have been either taken up less efficiently by the two invertebrates, con-
verted to nonachlors, or preferentially metabolized and excreted. The
relationships may also be altered to some extent by the aqueous concentra-
tion since there were notable diminutions in the ratios of cis-/trans-nonach1or
and to a much lesser extent for cis-/trans-chlordane at the~hTghest insecti-
cide levels. It can be speculated that if metabolic processes had a role in
altering the proportions of the constituents in the tissues, uptake may have
been sufficient in the brief period of exposure to temporarily supercede
metabolic processes.
Obviously, these observations raise basic questions as to the rela-
tive uptake, metabolism, and excretion of these constituents, questions
which can only be resolved by additional study. Such investigations are
beneficial since the stored compounds may have different toxicological
properties which would underlie any potential effects on predators. Labora-
tory studies of bioaccumulation should also be integrated with controlled
experiments in semi-natural environments and with sampling of aquatic organ-
isms in natural environments in order to verify the laboratory results.
80
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89
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APPENDIX TABLES
No. Page
1 Approximate Composition of Technical
Chlordane as Reflected in "Normalized"
Chromatogram of Significant Peaks 92
2 Representative Quality of Laboratory
Water 93
3 Standard Concentration-Percent Mortality
Data Supplied by Committee on Methods
For Toxicity Tests with Aquatic Organisms 94
4 Comparison of Median Lethal Concentrations
and 95% confidence Limits With Other
Aquatic Toxicology Laboratories 95
5 Median Lethal Concentrations (LC50) for
Daphm'a magna and Hyallela azteca Exposed
to Technical Chlordane 96
6 Median Lethal Concentrations (LC50) for
Fathead Minnow Juveniles Exposed to
Technical Chlordane 97
7 Median Lethal Concentrations (LC50) for
Brook Trout Exposed to Technical
Chlordane 98
8 Median Lethal Concentrations (LC50) for
Bluegill Exposed to Technical Chlordane 99
9 Significance of Differences in 96-Hr
LC50 Between Daphm'a magna. Fathead
Minnows, Brook Trout, and Bluegill 99
10 Water Quality During Exposure of
Fathead Minnows to Chlordane 100
90
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No. Page
11 Measured Technical Chlordane Concentra-
tions During Chronic Toxicity Test Using
Fathead Minnows 104
12 Water Quality During Chronic Exposure of
Bluegill to Technical Chlordane 105
13 Technical Chlordane Concentrations During
Chronic Toxicity Test Using Bluegill 110
14 Water Quality During Chronic Toxicity
Test of Technical Chlordane Utilizing
Brook Trout Ill
15 Measured Concentrations of Technical
Chlordane in Chronic Toxicity Test
Utilizing Brook Trout 116
16 Water Quality During Chronic Exposure
of Myall el a azteca to Technical
Chlordane 118
17 Measured Concentrations of Technical
Chlordane in Chronic Toxicity Test
Utilizing Hyallela azteca 119
18 Analysis of Variance of Wet Body Weights,
Dry Body Weights and (^[l-Chlordane Con-
tents of Myall ela azteca Exposed to
Different Concentrations of Technical
Chi ordane 120
19 Water Quality During Chronic Exposure of
Daphnia magna to Technical Chlordane 121
20 Measured Concentrations of Technical
Chlordane in Chronic Toxicity Test Using
Daphnia magna 122
21 Water Quality During Chronic Toxicity
Tests Utilizing Chironomus No. 51 123
22 Measured Concentrations of Technical
Chlordane in Chronic Toxicity Test
Utilizing Chironomus No. 51 125
91
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APPENDIX TABLE 1. APPROXIMATE COMPOSITION OF TECHNICAL
CHLORDANE AS REFLECTED IN "NORMALIZED" CHROMATOGRAM OF
SIGNIFICANT PEAKS3
Constituent Percentage
C10H7C15 " D1els-A1der Adduct (DAA): Penta-
chlorocyclopentadiene and
cyclopentadiene (C5Hg; "Cyclo") 2 ^ 1%
C^gHgClg - Isomers 1n order of GLC retention
time
(1} Isomer-1, chlordane - DAA;
hexachlorocyclopentadlene
(Hex) and *Cyclo" 1 ± 1%
(2) Isoraer-2 7.5 + 2%
(3) Isomers-3, 4 (combined) 13 +_ 2*
C10H5C17 " HePtachl<>r 10 1 3%
C10H6C18 ~ Chlordane ^somers
(1) cj[s_-chlordane 19^3$
(2) trans-chlordane 24 + 2%
C10H5C19 " Nonachlor 7 + 3X
Other constituents:
Hex (C5C16) Maximum lit
Octachlorocyclopentene 1 +_ IX
C10H7-8C16-7 8-5 ± 2*
Constituents of lower GLC retention
time than CgClg, including Hex 2^2%
Constituents of higher GLC retention
time than nonachlor 4^3%
aThe foregoing approximations are based upon unadjusted values
derived from moderate resolution gas-liquid chromatography.
Apparent values obtained are typically influenced by conditions
of analysis and the chromatographic systems employed, and the
relative response sensitivity of the components. Under stand-
ardized conditions, these profiles are useful in comparing
Technical Chlordane samples with Reference Technical Chlordane
(taken from Velslcol Chemical Corp. [12]).
92
-------
APPENDIX TABLE 2. REPRESENTATIVE QUALITY OF LABORATORY WATER
Variable
Calcium
Magnesium
Potassium •
Sodium
Chloride
Sulfate
Sulfide
Nitrate
Nitrite
Ammonia
Phenol
Fluoride
Unit
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
NHo-N
3
mg/1
mg/1
Mean
concentration
31.1
13.1
2.0
15.4
11.3
8.6
<0.002
4.65
0.005
0.16
0.001
0.96
Variable
Cyanide
Iron
Copper
Zinc
Cadmi urn
Chromium
PH
Alkalinity
Acidity
Total hard-
ness
Specific
conductance
Unit
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
CaCO,
o
mg/1
CaC03
mg/1
CaC03
umhos/
cm
Mean
concentration
0.0005
0.001
0.005
0.001
0.010
0.025
7.70
166
6
156
376
-------
APPENDIX TABLE 3. STANDARD CONCENTRATION-PERCENT
MORTALITY DATA SUPPLIED BY COMMITTEE ON METHODS FOR
TOXICITY TESTS WITH AQUATIC ORGANISMS3
Percent mortality
Toxicant concentration,
Data set
A
B
C
D
E
Control
0
0
0
0
0
7.8
0
0
0
0
0
13
0
0
0
0
0
22
10
70
10
20
20
36
100
100
40
70
30
yg/l
60
100
100
100
100
100
100
100
100
100
100
100
aData used to check the validity of statistical analyses of
acute toxicity test results. Ten specimens/treatment.
94
-------
APPENDIX TABLE 4. COMPARISON OF MEDIAN LETHAL CONCENTRATIONS AND 95% CONFIDENCE LIMITS
WITH OTHER AQUATIC TOXICOLOGY LABORATORIES3
to
l/l
Our laboratory
Litchfield
and Wilcoxon (64)
Computer program
Average of eight
other laboratories
Data
set
A
B
C
D
E
LC50, 95% confidence
yg/1 limits for LC50
25.4 22.1 - 29.2
21.2 18.8 - 23.9
35.5 28.2 - 44.7
29.8 23.9 - 37.2
35.5 27.1 - 46.9
LC50, 95% confidence LC50,C
jjg/1 limits for LC50 yg/1
d 25 3
• • • • • • C-+J • O
+1.9
• • • • • • tU» *f
+0.9
35.6 30.3 - 41.7 35.3
+2.4
29.5 25.0 - 34.7 29.4
+0.8
35.5 29.1 - 43.3 36.5
+3.4
95% confidence
limits for LC50
20.4
+5.1
14.8
+4.0
27.5
+3.3
23.5
+1.9
28.2
+.3.5
- 32.0
+4.8
- 49.3
+57.0
- 45.1
±3.6
- 36.9
+1.2
- 46.8
+4.3
aAll calculations performed on standard data given in Appendix Table 3.
bResults obtained through use of both manual and computer methods.
cMeans +_ 1 standard deviation are given for concentrations in pg/1.
Calculations not made since computer was not programmed to process data having only one
partial kill.
-------
APPENDIX TABLE 5. MEDIAN LETHAL CONCENTRATIONS (LC50) FOR
DAPHNIA MAGNA AND HYALLEJLA AZTECA EXPOSED TO TECHNICAL
CHLORDflNE
95% confidence
Exposure LC50, limits for LC50, Log-probit
L
time, hr yg/1 ug/1 a regression equation
D. magna
70
96
D. magna
66C
74C
94C
Myall el a
168
- Test 1
31.1
28.4
- Test 2
42.2
37.5
35.2
azteca
97.1
27.1
25.3
39.0
34.0
30.1
70.9
- 35.7
- 31.9
- 45.7
- 41.3
- 41.2
- 133.0
0.1594 -4.37+6.28 (log x..)
0.1329 -5.94+7.53 (log x^
1.1121
1.1726
1.2910
0.4056 0.10+2.47 (log x^
logarithm of the standard deviation of the population tolerance
frequency distribution.
bProbit yi = a + b (log x..).
Calculated according to the method of Litchfield and Wilcoxon (64).
The slope function S replaces a, and is the antilogarithm of
o.
96
-------
APPENDIX TABLE 6. MEDIAN LETHAL CONCENTRATIONS (LC50) FOR
FATHEAD MINNOW JUVENILES EXPOSED TO TECHNICAL
CHLORDANE
Exposure
time, hr
45C
72
96
120
168
192
LC50,
yg/1
53.4
41.7
36.9
35.9
33.9
32.1
95% confidence
limits for LC50, ^a
yg/1 cr
* • •
38.3 - 45.5
33.0 - 41.3
32.6 - 39.5
30.5 - 37.6
29.5 - 35.0
...
0.1022
0.1301
0.112
0.1175
0.0956
Log-probit
regression equation
...
-10.86+9.79 (log x^
- 7.27+7.69 (log x.)
- 9.11+9.08 (log x^
- 8.03+8.51 (log x..)
-10.76+10.46 (log x/
aLogarithm of the standard deviation of the population tolerance
frequency distribution.
bProbit y1 = a + b (log x^.
cMedian lethal time.
97
-------
APPENDIX TABLE 7. MEDIAN LETHAL CONCENTRATIONS (LC50) FOR
BROOK TROUT EXPOSED TO TECHNICAL CHLORDANE
Exposure
time, hr
27. 2C
28. 9C
46
96d
118
142
166
95% confidence
LC50, limits for LC50, a Log-probit
*> h
yg/l yg/l a regression equation
• £3 • • • ••• •»•
117
102 93-112 0.0681 -24.49+14.68 (log x^
*( ••• ••• • • *
39 34 - 44 0.0450 -30.30+22.21 (log x^
31 26 - 38 0.1494 15,05+6.69 (log x..)
25 21 - 29 0.1192 18.49+8.39 (log x^
Logarithm of the standard deviation of the population tolerance
frequency distribution.
5 A
Probit y.j = a + b (log x..).
:Med1an lethal time.
Interpolated from regression equation; (LC50) calculated from known
LC50 values and exposure times.
98
-------
APPENDIX TABLE 8. MEDIAN LETHAL CONCENTRATIONS (LC50) FOR
BLUEGILL EXPOSED TO TECHNICAL CHLORDANE
Exposure LC50,
95% confidence
limits for LC50,
time, hr ug/1 yg/1
48
72
96
120
144
121
77
59
46
40
98
68
50
39
35
- 149
- 87
- 71
- 54
- 45
o
0.2051
0.1397
0.2057
0.1759
0.1233
Log-probit
regression equation
-5.15+4.88 (log
-8.50+7.16 (log
-3.62+4.86 (log
-4.40+5.65 (log
-7.99+8.11 (log
xi}
xi}
*i>
xi)
xi>
Logarithm of the standard deviation of the population tolerance
frequency distribution.
. A
Probit y. = a + b (log x.).
APPENDIX TABLE 9. SIGNIFICANCE OF DIFFERENCES IN
96-HR LC50 BETWEEN DAPHNIA MAGNA, FATHEAD MINNOWS,
BROOK TROUT, AND COEG~ILL
Comparison
13. magna vs fathead minnow
Fathead minnow vs brook trout
Brook trout vs bluegill
D. magna vs brook trout
D_. magna vs bluegill
Fathead minnow vs bluegill
t-value
-1.754
-1 .232
-1.094
-3.520
-2.989
-1.651
Significance
N.S.
N.S.
N.S.
p<0.001
p<0.001
N.S.
99
-------
APPENDIX TABLE 10. WATER QUALITY DURING EXPOSURE OF FATHEAD MINNOWS TO CHLORDANE
o
o
Date
12/11/72
12/18/72
12/25/72
1/1/73
1/8/73
1/15/73
1/22/73
1/29/73
2/5/73
2/12/73
2/19/73
2/26/73
3/5/73
Water
temperature,
°C
21.5 +_ 0.6
22.5 +_ 0.9
23.8 +_0.9
23.2 +_0.5
24.0 +_ 1.9
24.1 +2.3
23.4 + 1.2
24.7 +0.4
24.5 +.0.7
24.9 + 0.2
25.0 + 0.3
25.0 +0.4
25.3 +0.2
Dissolved
oxygen,
mg/1 saturation
9.0
8.2
8.7
8.6
8.5
7.6
7.5
7.8
7.5
7.7
7.4
...
7.4
102.1
93.0
99.2
101.5
95.9
92.3
88.4
92.8
88.0
91.1
87.8
...
88.6
pH
7.81
7.80
8.18
7.94
7.91
7.93
7.79
7.86
7.88
7.79
7.80
7.68
7.67
Total
alkalinity,
mg/1 CaCOq
162
158
164
166
165
165
168
166
163
163
157
162
168
Total Specific
hardness, conductance,
mg/1 CaCOg ymhos/cm
163 ...a
* • • • * t
168
157
155
159
173
160
161
1 58
155
163 368
172 337
Continued ...
-------
APPENDIX TABLE 10. WATER QUALITY DURING EXPOSURE OF FATHEAD MINNOWS TO CHLORDANE—continued
Date
3/12/73
3/19/73
3/26/73
4/2/73
4/9/73
4/16/73
4/23/73
4/30/73
5/7/73
5/14/73
5/21/73
5/28/73
6/4/73
Water
temperature,
0 C
25.0 +0.3
24.8 + 0.8
24.4 + 1.4
25.1 + 0.2
25.2 +_ 0.3
25.3 + 0.2
25.1 + 0.7
25.3 + 0.3
25.3 +0.2
25.1 +0.4
24.9 +0.2
25.6 + 0.1
25.3 + 0.2
Dissolved
oxygen ,
%
mg/1 saturation
6.6
6.4
6.8
6.8
6.9
6.2
6.4
6.2
6.8
5.7
6.1
6.0
6.1
77.5
75.7
80.4
81.2
81.9
74.1
77.8
74.1
81.4
68.1
72.0 -
72.8
73.5
pH
7.70
7.58
7.60
7.62
7.50
7.47
7.50
7.60
7.70
7.45
7.52
7.73
7.48
Total
alkalinity,
mg/1 CaC03
163
163
163
167
161
166
160
159
162
166
162
160
158
Total
hardness,
mg/1 CaC03
158
164
167
146
142
158
148
143
145
161
152
160
147
Specific
conductance,
ymhos/cm
360
377
327
383
360
385
380
360
• • •
410
385
400
370
Continued ....
-------
APPENDIX TABLE 10. WATER QUALITY DURING EXPOSURE OF FATHEAD MINNOWS TO CHLORDANE—continued
o
to
Date
6/11/73
6/18/73
6/25/73
7/2/73
7/9/73
7/16/73
7/23/73
7/30/73
8/6/73
8/13/73
8/20/73
8/27/73
9/3/73
Water
temperature,
°C
24.9 + 0.1
24.8 + 0.1
24.8 + 0.2
24.7 + 0.4
24.4 + 0.5
24.4 + 0.3
24.9 +_ 0.4
25.2 +0.1
24.9 +0.2
24.9 +0.1
24.9 + 0.1
24.5 +0.1
24.3 +0.1
Dissolved
oxygen,
%
mg/1 saturation
5.9
6.0
5.8
6.5
6.6
6.4
6.5
6.6
6.6
6.6
6.7
7.3
7.4
82.0
71.1
69.9
73.0
77.7
75.7
77.6
78.1
78.6
79.9
79.4
86.9
88.1
PH
7.55
7.73
7.74
7.60
7.72
7.64
7.57
7.62
7.57
7.65
7.67
7.73
7.65
Total
alkalinity,
mg/1 CaCOg
* • •
169
165
169
168
172
172
170
176
174
172
169
173
Total
hardness,
mg/1 CaCOj
162
153
137
152
147
154
160
152
162
159
157
155
162
Specific
conductance,
ymhos/cm
400
388
337
351
354
375
398
379
398
408
339
376
404
Continued ....
-------
APPENDIX TABLE 10. WATER QUALITY DURING EXPOSURE OF FATHEAD MINNOWS TO CHLORDANE—continued
o
CrJ
Water
temperature,
Date °C
9/10/73 24.6 + 0.3
9/24/73 24. 3 +.0.1
10/1/73 24.6 + 0.3
10/8/73 24. 3 +_ 0.1
Mean
Standard
deviation
No. of . ...
observations
Dissolved
oxygen ,
%
mg/1 saturation
6.9
7.0
6.9
• * •
6.9
0.8
237
87.3
82.9
82.2
• * •
82.5
8.7
237
PH
7.67
7.88
7.70
7.89
7.70
0.15
180
Total
alkalinity,
mg/1 CaC03
168
165
170
163
166
5
180
Total
hardness,
mg/1 CaCOj
160
145
166
148
156
8
178
Specific
conductance,
ymhos/cm
375
365
405
389
376
23
61
aNo observation. Majority of later conductance readings were composites collected from control,
mid-range, and high chlordane concentrations.
b
Monitored continuously.
-------
APPENDIX TABLE 11. MEASURED TECHNICAL CHLORDANE
CONCENTRATIONS DURING CHRONIC TOXICITY TEST USING FATHEAD
MINNOWS
Sample
date
3/22/73
4/6/73
4/11/73
4/16/73
4/24/73
4/30/73
5/7/73
5/18/73
5/24/73
5/30/73
6/8/73
6/13/73
6/20/73
7/3/73
7/11/73
7/18/73
7/26/73
8/16/73
8/30/73
9/6/73
9/13/73
9/17/73
9/27/73
1 0/4/73
1 0/9/73
Mean
Standard
deviation
No. of
Desired concentration
Control
0.010
0.120
0.010
0.066
Tra
Tr
0.082
0.039
Tr
0.140
Tr
Tr
0.080
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
• • *
24
0.625
0.661
0.490
0.246
0.260
0.341
0.238
0.190
0.397
0.226
0.552
0.362
0.338
0.198
0.444
0.557
0.526
0.514
0.072
0.136
0.246
0.311
0.614
0.437
0.2gO
• • *
0.360
0.159
24
1.25
1.050
0.530
0.678
0.512
0.646
0.585
0.678
0.755
0.684
0.716
0.728
0.429
1.500
0.906
0.836
0.960
0.854
0.4J7
•
•
•
•
*
•
*
0.749
0.254
18
of chlordane, yg/1
2.50
1.520
1.820
0.930
0.794
1.720
0.860
0.214
1.530
1.290
1.660
2.340
1.240
0.264
1.750
1.910
1.800
1.590
1.5 JO
• * •
• • •
• • •
• • •
• • •
• • •
• » •
1.375
0.570
18
5.0
3.150
2.730
2.960
1.840
3.280
1.750
0.358
3.520
3.460
3.590
3.950
2.380
0.322
3.300
3.830
3.880
3.060
2.62.0
• • *
2.840
• • •
• • •
4.360
2 '.620
2.780
1.058
21
10.0
6.830
6.460
4.840
3.490
6.970
3.450
0.888
5.790
7.070
2.920
7.710
4.500
\J
• • •
7.420
6.380
7.690
7.000
6.440
5.4gO
***c
w
• • *
8.580
11.4JO
5*. 280
6.027
2.248
21
observations
aTrace amounts of less than 5 ng/1 chlordane detected.
bNo observation.
cNo measurements made because of no fish in test container.
104
-------
APPENDIX TABLE 12. WATER QUALITY DURING CHRONIC EXPOSURE OF BLUEGILL TO TECHNICAL CHLORDANE
o
Ul
Date
12/12/728
12/20/72
12/27/72
1/3/73
1/10/73
1/19/73
1/24/73
1/31/73
2/7/73
2/14/73
2/21/73
2/28/73
3/7/73d
Water
temperature,
°C
26.5 + 0.4b
19.4 + 0.5
19.5 + 0.7
19.1 +0.5
19.2 + 0.9
19.3 + 0.5
18.8 +_ 0.6
20.4 + 0.1
20.2 + 0.
20.1 +_ 0.1
20.2 + 0.1
20.1 +0.1
20.6 + 0.
Dissolved oxygen,
mg/1
5.0
6.2
5.5
6.5
5.6
6.3
5.6
5.9
4.9
5.5
5.6
5.2
4.8
%
saturation
61.2
67.7
59.9
70.2
61.1
60.4
60.4
63.0
53.2
60.0
52.6
56.7
51.8
PH
7.65
7.46
7.54
7.58
7.55
7.60
7.50
7.53
7.48
7.54
7.49
7.40
7.32
Total
alkalinity,
mg/1 CaC03
164
155
167
164
170
166
166
165
165
157
167
160
168
Total Specific
hardness, conductance,
mg/1 CaCOo ymhos/cm
154 ...c
173
171
157
157
157
161
159
164
156
167
• • * • • •
1 66 380
Continued ....
-------
APPENDIX TABLE 12. WATER QUALITY DURING CHRONIC EXPOSURE OF BLUEGILL TO TECHNICAL CHLORDANE
continued
o
ON
Date
3/14/73
3/19/73
3/21/73
3/28/73
3/31 /73e
4/4/73
4/11/73
4/18/73
4/25/73f
4/26/73f
4/30/73
5/2/73
5/9/73
Water
temperature,
°C
20.4 i 0.2
20.3 + 0.2
20.1 +0.1
20.1 + 0.1
• • •
21.1 + 1.2
23.9 + 0.5
23.8 + 1.6
25.0 +0.5
• • •
26.7 + 1.1
• • *
27.6 +_ 0.3
Dissolved
oxygen ,
%
mg/1 saturation
5.3
5.0
4.7
4.3
7.0
6.3
6.3
5.8
4.0
4.6
6.3
5.5
5.8
57.5
54.7
50.6
46.9
81.5
68.6
85.2
67.8
48.5
54.4
78.9
69.2
69.6
PH
7.43
* • •
7.26
7.32
• k .
7.37
7.52
7.50
• * •
7.32
• » •
7.62
7.73
Total
alkalinity,
mg/1 CaC03
163
• • •
163
164
• • •
168
160
166
• • »
161
• • •
163
162
Total
hardness,
mg/1 CaC03
166
• * *
172
171
• • •
155
143
158
• • •
146
• * •
159
146
Specific
conductance,
vimhos/cm
353
• • *
353
377
• » •
380
362
360
t • •
370
• • •
• • *
393
Continued ....
-------
o
•-o
APPENDIX TABLE 12. WATER QUALITY DURING CHRONIC EXPOSURE OF BLUEGILL TO TECHNICAL CHLORDANE
continued
Date
5/17/73
5/23/73
5/31/73
6/6/73
6/13/73
6/20/73
6/27/73
7/3/73
7/10/73
7/17/73
7/24/73
7/31/73
8/6/73
Water
temperature,
°C
28.0 +0.2
28.1.+ 0.3
28.2 + 0.2
28.2 + 0.3
28.0 +0.1
27.9 +.0.1
28.0 +_ 0.1
27.9 + 0.2
27.8 +_ 0.1
27.7 + 0.1
27.9 + 0.1
28.0 + 0.1
27.7 +0.1
Dissolved
oxygen ,
%
mg/1 saturation
6.0
5.7
6.5
5.1
6.2
5.9
6.5
5.8
6.6
6.1
6.7
6.4
6.8
76.0
72.6
81.6
64.7
78.1
74.1
82.1
73.8
83.3
76.6
85.0
80.5
84.0
PH
7.73
7.53
7.91
7.54
7.74
7.82
8.02
7.73
7.85
7.78
7.94
7.70
7.84
Total
alkalinity,
mg/1 CaC03
164
161
155
160
161
164
175
167
167
166
167
170
173
Total
hardness,
mg/1 CaC03
155
143
155
146
147
153
159
145
148
141
146
152
153
Specific
conductance,
ymhos/cm
420
387
• • •
385
370
390
395
375
370
375
380
400
390
Continued ..„.
-------
APPENDIX TABLE 12. WATER QUALITY DURING CHRONIC EXPOSURE OF BLUEGILL TO TECHNICAL CHLORDANE
continued
o
oo
Date
8/13/73
8/20/73
8/27/73
9/4/73
9/11/73
9/18/73
9/25/73
10/2/739
10/9/73
10/16/73
11/23/73
Water
temperature,
°C
27.9 + 0.2
28.0+0.1
27.7 + 0.1
27.7 +0.1
27.9 +0.2
27.8 +0.1
27.4 + 0.2
27.6 +0.2
27.4 +0.1
27.9 +0.1
27.9 +0.1
Dissolved
oxygen ,
mg/1 saturation
6.1
5.6
6.1
6.4
5.8
...
6.6
7.0
6.9
6.3
6.9
77.2
70.9
77.2
80.6
69.1
...
82.8
88.3
87.5
80.1
87.4
PH
7.74
7.71
7.69
7.79
7.72
* • •
7.91
7.93
7.98
7.80
7.92
Total
alkalinity,
mg/1 CaC03
173
173
169
173
169
...
165
169
161
166
159
Total
hardness,
mg/1 CaC03
159
157
154
159
158
...
144
162
145
157
146
Specific
conductance,
ymhos/cm
395
385
385
394
371
...
368
390
386
386
368
Contlnued ....
-------
o
to
APPENDIX TABLE 12. WATER QUALITY DURING CHRONIC EXPOSURE OF BLUEGILL TO TECHNICAL CHLORDANE
continued
Water
temperature,
Date °C
Mean 24. 7 ±3. 8
Standard ...
deviation
No. of 267
observations
Dissolved oxygen,
mg/1
5.9
0.7
279
%
saturation pH
69.9 7.65
11.8 0.20
279 184
Total
alkalinity,
mg/1 CaC03
165
5
184
Total
hardness,
mg/1 CaC03
156
9
184
Specific
conductance,
ymhos/cm
380
14
31
Dissolved oxygen determined with Yellow Springs Instrument Company oxygen meter (Model No. 54).
Values given are means, except for water temperature where mean +_ 1 standard deviation is given.
°No observation.
Dissolved oxygen determined with azide modification of Winker method (21).
Artificial aeration instituted.
Malfunction of aeration equipment.
gMean acidity from 10/2/73 to 11/23/73 was 4.42 mg/1 CaCO- with a standard deviation of 2.44.
-------
APPENDIX TABLE 13. TECHNICAL CHLORDANE CONCENTRATIONS
DURING CHRONIC TOXICITY TEST USING BLUEGILL
Sample
date
3/26/73a
3/20/73
4/5/73
4/10/73
4/16/73
4/25/73
4/30/73
5/7/73
5/18/73
5/29/73
6/4/73
6/6/73
6/13/73
6/19/73
7/2/73
7/11/73
7/18/73
7/27/73
8/1/73
8/17/73
8/30/73
9/6/73
9/14/73
9/21/73
9/27/73
10/4/73
10/9/73
10/19/73
Mean
Standard
deviation
Nominal concentration of chlordane, yg/1
Control
0.08
0.06
0.05
0.00
0.07
0.01
0.00
0.00
0.09
0.02
0.02
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.03
0.625
0.06
0.26
0.05
0.25
0.26
0.19
0,24
0.29
0.30
0.28
0.38
0.00
0.17
0.34
0.32
0.33
0
...
0.33
...
o.n
0.19
0.21
0.51
0.31
0.37
0.44
0.15
0.26
0.25
0.12
1.25
0.93
0.87
0.22
0.23
0.46
0.46
0.26
0.53
0.61
0.53
0.34
0.17
0.56
0.52
0.73
0.74
0
...
0.78
0.35
0.43
D
• • *
0.48
0.64
0.42
0.72
0.83
0.57
0.60
0.54
0.21
2.5
0.72
0.94
0.36
1.07
1.02
1.16
0.85
1.98
1.70
1.34
0.80
1.16
1.30
1.29
1.23
1.43
0.39
1.43
1.22
1.55
0.66
0.42
1.20
1.25
2.90
1.85
1.49
1.47
1.22
0.53
5
1.95
2.10
1.00
2.86
2.34
2.38
1.43
2.12
2.71
2.51
1.98
1.95
2.81
2.44
2.27
270
./»
0
...
2.90
2.19
2.00
0.65
1.73
2.28
2.88
D
• • •
2.36
D
...
2.48
2.20
0.56
10
2.52
5.84
2.58
3.75
4.16
5.24
3.56
4.76
5.98
6.32
4.49
4.86
6.64
6.69
4.83
7-¥
U
* • •
7.77
4.37
5.36
3.44
4.18
8.88
6.15
...b
5.32
D
• • •
4.11
5.17
1.57
aBecause of analytical problems, data prior to this date were not
considered reliable, and consequently not entered.
bOther hexane-soluble substances encountered in water samples
which Interferred with chlordane measurement.
110
-------
APPENDIX TABLE 14. WATER QUALITY DURING CHRONIC TOXICITY TEST OF TECHNICAL CHLORDANE
UTILIZING BROOK TROUT
Date
3/30/733
4/3/73
4/10/73
4/16/73
4/23/73
4/30/73
5/8/73
5/14/73
5/21/73
5/29/73
6/5/73
6/ll/73C
6/18/73d
6/25/73
Water
temperature,
°C
10.8 + 0.5b
11.6 + 0.5
10.2 +1.5
11.3 + 1.5
12.1 +0.8
12.9 +_ 0.7
12.8 +0.3
13.2 +_ 0.5
14.1 + 0.6
14.3 +_0.2
14.8 + 0.3
14.8 + 0.1
14.6 +_ 0.2
14.2 + 0.1
Dissolved oxygen
mg/1
7.6
7.6
7.8
7.9
8.7
8.9
9.0
8.9
7.0
6.3
6.4
8.0
7.2
6.9
saturation
68.3
68.5
67.1
69.9
83.4
82.5
85.1
84.2
66.8
60.9
62.3
78.6
70.2
67.3
PH
7.37
7.10
7.10
7.25
7.50
7.48
7.60
7.40
7.37
7.69
7.32
7.52
7.61
7.71
Total
alkalinity, Acidity,
mg/1 CaC03
164
163
162
164
160
165
162
164
162
159
158
• • • • • •
166
164
Total
hardness.
143
164
154
157
148
158
148
160
149
160
148
160
151
141
Specific
conductance,
ymhos/cm
• • •
370
375
368
370
370
400
395
390
405
375
390
385
360
Lontinuea
-------
APPENDIX TABLE 14. WATER QUALITY DURING CHRONIC TOXICITY TEST OF TECHNICAL CHLORDANE
UTILIZING BROOK TROUT--cont1nued
IS)
Date
7/2/73
7/9/73
7/16/73
7/24/73
7/30/73
8/6/73
8/15/73
8/21/73
8/27/73
9/4/73
9/10/73
9/18/73
9/25/73
Water
temperature,
°C
14.0 +.0.2
14.5 +.0.4
14.4 +0.1
14.3 +0.6
14.5 +0.8
14.4 +0.1
14.8 + 0.1
15.1 +_0.7
15.0 +_0.4
13.7 + 1.0
13.1 +0.3
11.8 +_0.4
12.0 +0.3
Dissolved
oxygen
%
mg/1 saturation
7.5
7.4
8.0
8.1
8.0
8.5
8.3
8.0
8.7
8.1
8.5
9.0
8.4
73.3
71.4
77.1
79.6
75.8
82.5
81.7
78.8
85.7
77.6
79.5
82.9
78.2
PH
7.47
7.50
7.68
7.71
7.65
7.66
7.70
7.71
7.70
7.64
7.73
7.69
7.84
Total
alkalinity, Acidity,
mg/1 CaC03
168
170
171
168
170,
175
165
172
171
173
167
165 11.0
166 6.8
Total
hardness,
153
154
154
148
152
162
145
158
156
162
156
150
144
Specific
conductance,
umhos/cm
380
375
385
380
390
400
385
390
380
402
373
379
372
Continued ...
-------
APPENDIX TABLE 14. WATER QUALITY DURING CHRONIC TOXICITY TEST OF TECHNICAL CHLORDANE
UTILIZING BROOK TROUT—continued
Date
10/3/73
10/11/73
10/18/73
10/23/73
10/30/73
11/5/73
11/12/73
11/19/73
11/26/73
12/3/73
12/10/73
12/17/73
12/24/73
Water
temperature,
°C
10.8 +_ 0.7
12.0 + 2.6
10.4 + 0.6
10.6 +0.3
9.4 + 1.3
10.0 + 0.8
10.2 +0.4
10.2 + 0.5
10.7 + 1.3
10.2 +_0.3
10.2 + 0.2
10.1 +_ 0.1
10.2 + 0.2
Dissolved oxygen
Total
„ alkalinity, Acidity,
A)
mg/1
9.5
9.0
9.4
9.0
9.5
10.0
8.8
9.2
8.6
9.8
9.6
9.6
9.9
saturation
83.8
83.3
83.6
81.5
82.2
87.7
78.9
82.2
75.2
87.1
85.7
85.0
87.8
pH
7.87
7.70
7.63
7.70
7.76
7.80
7.88
7.66
7.82
8.15
7.82
8.04
8.53
168
159
165
166
159
163
166
143
145
153
143
145
158
mg/1 CaC03
• • •
10.3
6.8
6.3
4.5
6.0
11.1
4.5
14.3
4.4
8.0
7.1
• • •
Total
hardness,
164
143
160
157
145
152
159
140
141
160
143
133
139
Specific
conductance,
ymhos/cm
405
386
397
396
379
387
411
• • •
374
• • •
377
357
375
Continued ....
-------
APPENDIX TABLE 14. WATER QUALITY DURING CHRONIC TOXICITY TEST OF TECHNICAL CHLORDANE
UTILIZING BROOK TROUT -continued
Date
12/31/73
1/7/74
1/14/74
1/21/74
1/28/74
2/4/74
2/11/74
2/18/74
2/25/74
3/4/74
3/11/74
3/18/74
3/25/74
Water
temperature,
t
9.6 +.0.4
9.7 +0.2
9.4 + 0.4
9.6 + 0.5
9.8 + 0.6
8.9 +_0.3
9.2 +0.4
9.7 + 0.4
10.3 +0.2
10.1 +0.4
10.5 +0.2
10.3 +.0.2
10.2 +0.2
Dissolved oxygen
Total
,, alkalinity, Acidity,
fO
mg/1
9.4
10.1
10.0
9.4
9.7
9.6
9.8
9.5
8.5
9.6
8.2
6.8
6.8
saturation
83.0
88.7
86.2
81.7
84.4
84.7
86.7
84.5
76.2
85.4
72.9
59.7
60.1
PH
7.83
8.00
7.96
7.83
7.82
7.87
7.83
7.79
7.86
7.92
7.83
7.87
7.73
155
158
146
140
144
155
155
156
151
153
158
158
156
mg/1 CaCO^
o
11.8
11.5
8.6
7.7
6.3
10.7
6.3
5.8
6.2
4.2
6.0
4.8
4.3
Total
hardness,
142
144
135
136
145
142
136
148
139
142
147
135
136
Specific
conductance,
umhos/cm
394
387
365
376
380
369
367
380
361
367
374
356
376
Continued ....
-------
APPENDIX TABLE 14. WATER QUALITY DURING CHRONIC TOXICITY TEST OF TECHNICAL CHLORDANE
UTILIZING BROOK TROUT—continued
Water
temperature,
Date °C
4/1/74 10
4/8/74 9
4/15/74 10
4/22/74 10
Mean
Standard
deviation
No. of
observations
.0 +0.3
.9 +0.1
.2 + 0.4
.8 +_ 0.6
...
...
419
Dissolved oxygen
Total
« alkalinity, Acidity,
mg/1
6.6
7.3
7.1
6.7
8.3
1.2
247
saturation
59.1
71.1
63.3
62.9
75.7
9.9
247
PH x
7.82
7.77
7.62
7.62
7.68
0.26
174
149
163
160
160
160
8
174
mg/1 CaCOo
3.5
5.4
7.3
6.8
7.3
2.7
79
Total
hardness,
134
162
160
157
150
10
174
Specific
conductance,
ymhos/cm
358
421
413
417
380
23
73
Dissolved oxygen determined with azide modification of Winkler method (21).
^Values are means. Standard deviations given for temperature data.
>%
"Artificial aeration instituted.
Dissolved oxygen determined with Yellow Springs Instrument Company Oxygen Meter after this date.
-------
APPENDIX TABLE 15. MEASURED CONCENTRATIONS OF
TECHNICAL CHLORDANE IN CHRONIC TOXICITY TEST
UTILIZING BROOK TROUT
Nominal chlordane concentration,
Date
4/9/73
4/11/73
4/16/73
4/24/73
5/1/73
5/7/73
5/18/73
5/24/73
6/4/73
6/6/73
6/13/73
6/19/73
7/3/73
7/11/73
7/18/73
7/26/73
8/1/73
8/16/73
8/30/73
9/6/73
9/13/73
9/21/73
9/27/73
10/4/73
10/9/73
10/19/73
10/26/73
11/16/73
1 1/23/73
11/30/73
12/14/73
12/21/73
12/28/73
1/2/74
1/8/74
1/15/74
1/25/74
0.625
0.43
0.42
0.34
0.35
0.03
0.36
0.34
0.28
0.22
0.22
0.24
0.42
D
* . .
0.31
0.18
0.33
0.10
0.09
0.23
0.14
0.41
0.24
0.31
0.37
0.41
0.25
0.37
0.45
0.28
0.32
0.21
0.20
0.25
0.28
0.40
0.30
0.32
1.25
0.77
0.64
0.92
0.49
0.06
0.76
0.66
0.59
0.97
0.55
0.66
0.57
1.28
1.24
0.87
0.63
0.92
0.17
0.67
0.49
0.35
0.42
• • •
0.49
0.43
0.30
0.45
0.94
0.65
0.76
0.56
0.31
0.48
0.55
0.63
0.63
0.69
2.5
1.84
1.84
2.36
1.68
0.16
1.27
1.32
1.34
1.72
1.57
1.59
1.28
1.49
1.14
• • •
1.14
1.22
0.50
0.%
0.90
0.36
0.72
1.55
1.11
1.23
0.90
0.84
1.39
1.58
1.27
0.96
0.59
0.99
0.90
1.18
1.34
0.91
5.0
2.84
3.18
4.01
1.99
0.23
2.28
1.97
2.14
3.28
2.48
3.44
2.05
3.14
1.57
1.39
3.54
3.11
1.44
1.28
2.78
0.90
1.48
3.96
1.25
1.11
0.15
1.86
2.24
2.35
1.82
1.83
1.03
1.75
1.72
2.14
2.42
2.71
ug/la
10.0
7.14
6.44
7.93
6.80
0.41
5.79
7.07
8.42
2.02
7.86
6.29
9.24
• • •
9.71
6.45
8.43
4.02
6.46
6.58
7.25
3.49
6.36
• » •
5.30
6.79
0.37
8.01
3.70
5.35
6.97
• • •
3.18
4.53
7.63
5.74
5.24
4.98
Continued .,
116
-------
APPENDIX TABLE 15. MEASURED CONCENTRATIONS OF
TECHNICAL CHLORDANE IN CHRONIC TOXICITY TEST
UTILIZING BROOK TROUT~continued
Nominal chlordane concentration, yg/la
Date
2/5/74
2/14/74
2/21/74
2/27/74
3/6/74
3/13/74
3/20/74
3/28/74
4/10/74
4/16/74
4/23/74
5/2/74
Mean
Standard
deviation
No. of
0.625
0.39
0.10
0.42
0.65
• • •
0.30
1.16
0.22
0.29
0.41
0.37
0.65
0.32
0.18
47
1.25
0.74
0.84
0.69
0.73
• • •
0.52
1.16
0.40
0.74
0.79
0.78
1.11
0.66
0.25
47
2.5
1.52
1.10
1.39
1.23
1.95
0.91
2.62
0.90
1.58
1.70
1.61
2.06
1.29
0.48
48
5.0
1.98
2.34
2.45
2.10
2.56
1.42
2.88
1.64
2.23
2.80
2.52
4.37
2.21
0.89
49
10.0
• • »
4.31
• * •
4.79
• • *
2.71
• * •
3.38
4.74
6.29
4.51
7.86
5.80
2.15
42
observations
aLess than 0.11 yg/1 chlordane measured in control at any
time during test.
No observation.
117
-------
APPENDIX TABLE 16. WATER QUALITY DURING CHRONIC EXPOSURE OF HYALLELA AZTECA TO TECHNICAL
00
CHLORDANE
Date
4/2/74
4/10/74
4/17/74
4/24/74
4/30/74
5/6/74
5/13/74
5/20/74
Mean
Standard
deviation
Water
temperature,
°C
15.4 + 0.6
15.3 +1.4
17.5 +0.3
17.5 +0.3
17.5 + 1.4
16.5 +0.4
16.9 +0.1
16.7 +0.2
16.7
+J.O
No. of 40
measurements
Dissolved oxygen
Total
% alkalinity, Acidity,
mg/1
7.6
8.0
7.7
7.7
7.6
7.3
7.3
6.9
7.5
+0.4
24
saturation
74.8
82.8
77.8
78.5
78.7
73.3
72.7
69.7
76.1
+4.3
24
pH
7.97
8.03
7.63
7.86
7.80
7.90
7.83
7.80
7.86
+0.13
24
151
154
162
151
146
150
152
152
152
+5
24
mg/1 CaCO,
•J
3.7
3.5
7.7
5.1
6.9
4.3
5.5
5.5
5.3
+1.5
24
Total
hardness,
135
142
161
149
151
148
150
150
148
+7
24
Specific
conductance,
umhos/cm
359
376
419
393
385
385
401
395
388
+18
8
-------
APPENDIX TABLE 17. MEASURED CONCENTRATIONS OF
TECHNICAL CHLORDANE IN CHRONIC TOXICITY TEST UTILIZING
HYLALLELA AZTECA
Nominal chlordane concentration,
Date
3/28/74
4/5/74
4/10/74
4/16/74
4/23/74
5/3/74
5/7/74
5/17/74
5/23/74
Mean
Standard
deviation
2.5
0.24
0.52
0.82
1.93
1.82
1.28
2.68
1.85
1.56
1.41
0.77
5
0.72
0.83
1.69
4.25
3.31
2.91
4.31
2.95
2.75
2.64
1.32
10
1.08
1.72
3.71
3.16
9.65
5.05
9.92
7.57
6.00
5.32
3.24
20
3.32
3.06
6.47
14.50
16.40
13.70
21.40
12.60
12.30
11.53
6.14
yg/la
40
5.77
6.46
13.99
19.20
33.50
26.40
28.80
25.45
25.10
20.52
9.85
aNo chlordane detected in control chambers, except on
4/10/74, when 0.21 yg/1 was measured.
119
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APPENDIX TABLE 18. ANALYSIS OF VARIANCE OF WET
BODY WEIGHTS, DRY BODY WEIGHTS AND CIS-CHLORDANE CONTENTS
OF HYALLELA AZTECA EXPOSED TO DIFFERENT CONCENTRATIONS
OF TECHNICAL CHLORDANE
Source of error
Wet body weights -
Among treatments
Within treatments
Total
Degrees of
freedom
replicate I
4
95
99
Sum of
squares
39.62
140.13
179.75
Mean square F
9.90 6.71a
1.47
Wet body weights - replicate II
Among treatments 4 61.55 15.38 7.39a
Within treatments 100 201.54 2.01
Total 104 263.09
Dry body weights - both replicates
Among treatments
Within treatments
Total
4
5
9
0.47
0.16
0.63
0.11
0.03
3.53b
Significant at p <0.005.
Significant at p <0.001.
120
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APPENDIX TABLE 19. WATER QUALITY DURING CHRONIC EXPOSURE OF DAPHNIA MAGNA TO TECHNICAL
tSJ
t-'
CHLORDANE
Water
Dissolved oxygen
temperature,
Date °C mg/1
5/6/74
5/13/74
5/20/74
5/28/74
Mean
Standard
deviation
No. of
observations
20.9
20.3
21.0
21.4
20.9
0.5
4
5.8
6.0
5.0
• t •
5.6
0.5
9
%
saturation
64.7
66.0
55.7
• • #
62.0
5.1
9
c
PH
8.03
8.03
8.00
8.10
8.03
0.06
12
Total
ilkalinity, Acidity,
160
162
165
160
162
3
12
mg/1 CaC03
3.3
4.2
4.2
2.4
3.5
0.8
12
Total
hardness,
162
167
169
161
165
4
12
Specific
conductance,
umhos/cm
416
440
440
413
428
15
4
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APPENDIX TABLE 20. MEASURED CONCENTRATIONS OF
TECHNICAL CHLORDANE IN CHRONIC TOXICITY
TEST USING DAPHNIA MAGNA
Nominal concentration of
Date
5/7/74
5/17/74
5/23/74
Mean
Standard
deviation
Control
Oa
0
0
0
0
6.1
1.7
1.7
1.8
1.7
0.1
12.1
1.5
3.1
2.8
2.5
0.9
technical chlordane, yg/1
24.2
5.5
5.9
7.3
6.2
1.0
48.5
8.1
12.1
15.0
12.1
3.6
96.9
13.5
19.0
32.2
21.6
9.6
a
No chlordane detected.
122
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APPENDIX TABLE 21. WATER QUALITY DURING CHRONIC TOXICITY TESTS UTILIZING
CHIRONOMUS NO. 51
to
Date
Test 1
5/6/74
5/13/74
5/20/74
5/28/74
Mean
Standard
deviation
Water
temperature ,
°C
24.0
24.0
23.6
23.1
23.8
0.3
No. of 9
observations
Test 2
6/6/74
6/11/74
24.7
24.6
Dissolved
oxygen
%
mg/1 saturation pH
6.8
7.1
6.3
a
• » •
6.7
0.4
9
7.1
7.0
80.3 7.93
82.3 7.97
75.0 7.93
... 7.83
79.2 7.94
3.6 0.05
9 12
84.3 7.90
82.2 7.99
Total
alkalinity,
Acidity,
Total
hardness,
mg/1 CaCOo
155
154
153
156
154
2
12
158
147
4.0
4.7
4.5
3.3
4.2
0.7
12
4.5
2.8
154
154
153
155
154
2
11
155
141
Specific
conductance,
umhos/cm
393
406
401
392
398
7
4
398
351
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APPENDIX TABLE 21. • WATER QUALITY DURING CHRONIC TOXICITY TESTS UTILIZING
CHIRONOMUS NO. 51—continued
Water
temperature,
Date
6/21/74
6/24/74
Mean
Standard
deviation
No. of
observations
°C
24.4
24.2
24.4
0.3
22
Dissolved oxygen
Total
% alkalinity, Acidity,
mg/1 saturation pH
6.9 80.3 7.77
• • • ... /.o/
7.0 82.3 7.90
0.1 2.0 0.10
9 9 18
Total
hardness,
mg/1 CaCO,
0
160
155
153
5
18
5.4
4.8
4.2
1.1
18
160
151
150
8
18
Specific
conductance,
ymhos/cm
394
380
380
21
4
No observation.
-------
APPENDIX TABLE 22. MEASURED CONCENTRATIONS OF TECHNICAL
CHLORDANE IN CHRONIC TOXICITY TEST UTILIZING
CHIRONOMUS NO. 51
Measured chlordane concentration, pg/1
Date
Test 1
5/3/74
5/7/74
5/17/74
Mean
Standard
deviation
Test 2
6/6/74
6/11/74
6/24/74
Mean
Standard
deviation
Tank 1
Oa
0
0
0
» • •
0
0
0
0
• • •
Tank 2
1.1
0.9
1.0
1.0
0.1
0.5
0.8
0.8 .
0.7
0.1
Tank 3
3.4
1.8
2.0
2.4
0.9
0.9
2.2
2.0
1.7
0.7
Tank 4
• * •
4.6
3.6
4.1
0.7
2.9
3.3
3.6
3.3
0.4
Tank 5
25.9
9.8
7.1
14.3
10.2
5.6
6.9
9.5
7.3
2.0
Tank 6
48.0
15.2
10.2
24.9
16.8
11.8
15.6
19.2
15.5
3.7
No technical chlordane detected.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-77-019
3. RECIPIENT'S ACCESSION-NO.
4'TIIa3TEDlNDTICHRONIC TOXICITY OF CHLOEDANE TO FISH
AND INVERTEBRATES
5. REPORT DATE
February 1977 issuing date
B. PERFORMING ORGANIZATION CODE
7.AUTHOR(S) Rick D. Cardwell, Dallas G. Foreman,
Thomas R. Payne, and Doris J. Wilbur
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Chemico Process Plants Co.-Envirogenics Systems
9200 East Flair Drive
El Monte, California 91734
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
Contract 68-01-0187
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Project - Final
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
is. ABSTRACT jhe acute and chronic toxicity of technical chlordane to bluegill (Lepomis
macrochirus), fathead minnow (Pimephales promelas), brook trout (Salvelinus
fontinalis), Daphnia magna, Hyallela azteca, and Chironomus No. 51 were determined
with flow-through conditions. The purpose was to estimate concentrations producing
acute mortality and those having no effect on the long-term survival, growth, and
reproduction of the various species. Whole body residues of technical chlordane
components were measured in the three invertebrate species at the end of the chronic
exposure tests.
Concentrations of technical chlordane causing 50% mortality in 96 hr were
36.9 yg/1 for fathead minnow, 47 yg/1 for brcok trout, and 59 yg/1 for bluegill,
while that causing 50% immobilization in the cladoceran, I), magna, was 28.4 yg/1.
The amphipod, II. azteca, was only slightly affected at 96 hr by the chlordane
concentrations tested, and the 168-hr EC50 was 97.1 yg/1. Acute mortality of midges
Chironomus No. 51, was not successfully evaluated.
With respect to the test conditions employed and life cycle stages evaluated,
the lowest concentrations of technical chlordane found to cause major chronic
effects were 0.32 yg/1 for brook trout, 1.22 yg/1 for bluegill, 1.7 yg/1 for midges,
11.5 yg/1 for amphipods, and 21.6 yg/1 for cladocerans
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Freshwater Fishes
Trout
Minnows
Toxicity
Invertebrates
Daphnia
Chlordane
Insecticide
Residues
Bluegill
Amphipod
Midge
Chronic
Acute
06F, T, C
IS. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
136
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
E*A Form 2220-1 (»-73)
126
•fcO.l 60VERNWIIT HINTING OfFKt 1977-757-056/5592 Region Ho. 5-11
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